U.S. patent number 11,230,604 [Application Number 15/909,540] was granted by the patent office on 2022-01-25 for antibodies against human cd38.
This patent grant is currently assigned to GENMAB A/S. The grantee listed for this patent is GENMAB A/S. Invention is credited to Michel De Weers, Paul Parren, Jan Van De Winkel, Tom Vink, Tim Walseth.
United States Patent |
11,230,604 |
De Weers , et al. |
January 25, 2022 |
Antibodies against human CD38
Abstract
Isolated monoclonal antibodies which bind to human CD38 and
related antibody-based compositions and molecules, are disclosed.
Also disclosed are pharmaceutical compositions comprising the
antibodies and therapeutic and diagnostic methods for using the
antibodies.
Inventors: |
De Weers; Michel (Houten,
NL), Walseth; Tim (Roseville, MN), Van De Winkel;
Jan (Zeist, NL), Vink; Tom (Alphen aan den Rijn,
NL), Parren; Paul (Odijk, NL) |
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB A/S |
Copenhagen |
N/A |
DK |
|
|
Assignee: |
GENMAB A/S (Copenhagen V,
DK)
|
Family
ID: |
1000006071561 |
Appl.
No.: |
15/909,540 |
Filed: |
March 1, 2018 |
Prior Publication Data
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|
|
Document
Identifier |
Publication Date |
|
US 20180298106 A1 |
Oct 18, 2018 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14990869 |
Jan 8, 2016 |
9944711 |
|
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13702857 |
Feb 2, 2016 |
9249226 |
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PCT/EP2011/059507 |
Jun 8, 2011 |
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61353082 |
Jun 9, 2010 |
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Foreign Application Priority Data
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Jun 9, 2010 [DK] |
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PA201000498 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/2878 (20130101); G01N 33/573 (20130101); A61K
45/06 (20130101); C07K 16/2896 (20130101); A61K
39/3955 (20130101); C07K 2317/51 (20130101); G01N
2333/91148 (20130101); C07K 2317/75 (20130101); C07K
2317/734 (20130101); C07K 2317/76 (20130101); C07K
2317/565 (20130101); C07K 2317/732 (20130101); C07K
2317/33 (20130101); C07K 2317/34 (20130101); C07K
2317/21 (20130101); C07K 2317/56 (20130101); C07K
2317/515 (20130101) |
Current International
Class: |
C07K
16/28 (20060101); A61K 45/06 (20060101); A61K
39/395 (20060101); G01N 33/573 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0798386 |
|
Oct 1997 |
|
EP |
|
89/08114 |
|
Sep 1989 |
|
WO |
|
92/01049 |
|
Jan 1992 |
|
WO |
|
94/17184 |
|
Aug 1994 |
|
WO |
|
96/16990 |
|
Jun 1996 |
|
WO |
|
98/16245 |
|
Apr 1998 |
|
WO |
|
98/16254 |
|
Apr 1998 |
|
WO |
|
98/50435 |
|
Nov 1998 |
|
WO |
|
99/62526 |
|
Dec 1999 |
|
WO |
|
00/06194 |
|
Feb 2000 |
|
WO |
|
00/40265 |
|
Jul 2000 |
|
WO |
|
02/06347 |
|
Jan 2002 |
|
WO |
|
02/32288 |
|
Apr 2002 |
|
WO |
|
03/080672 |
|
Oct 2003 |
|
WO |
|
2004/019915 |
|
Mar 2004 |
|
WO |
|
2004/035607 |
|
Apr 2004 |
|
WO |
|
2004/045512 |
|
Jun 2004 |
|
WO |
|
2004/058288 |
|
Jul 2004 |
|
WO |
|
2005/042019 |
|
May 2005 |
|
WO |
|
2005/044855 |
|
May 2005 |
|
WO |
|
2005/103083 |
|
Nov 2005 |
|
WO |
|
2006/088951 |
|
Aug 2006 |
|
WO |
|
2006/099875 |
|
Sep 2006 |
|
WO |
|
06/125640 |
|
Nov 2006 |
|
WO |
|
2006/125640 |
|
Nov 2006 |
|
WO |
|
2008/047242 |
|
Apr 2008 |
|
WO |
|
2011154453 |
|
Dec 2011 |
|
WO |
|
Other References
Zocchi et al., "Ligand-Induced Internalization of CD38 Results in
Intracellular Ca2+ Mobilization: Role of NAD+ Transport Across Cell
Membranes," FASEB J. 13(2):273-283 (1999). cited by applicant .
Antonelli et al., "Anti-CD38 autoimmunity in patients with chronic
autoimmune thyroiditis or Graves' disease," Clin. Exp. Immunol.,
126(3):426-431 (2001). cited by applicant .
Antonelli et al., "Autoimmunity to CD38 and GAD in Type I and Type
II diabetes: CD38 and HLA genotypes and clinical phenotypes,"
Diabetologia, 45(9):1298-1306 (2002). cited by applicant .
Ausiello et al., "CD38 ligation induces discrete cytokine mRNA
expression in human cultured lymphocytes," Eur J. Immunol.
25:1477-80 (1995). cited by applicant .
Barata et al., "The Role of Cyclic-ADP-Ribose-Signaling Pathway in
Oxytocin-Induced Ca2+ Transients in Human Myometrium Cells,"
Endocrinology 145(2):881-89 (2004). cited by applicant .
Barone et al., "A pivotal role for cADPR-mediated Ca2+ signaling:
regulation of endothelin-induced contraction in peritubular smooth
muscle cells," FASEB J. 16:697-05 (2002). cited by applicant .
Barthelemy et al., "Comprehensive analysis of the factors
contributing to the stability and solubility of autonomous human VH
domains," Journal of Biological Chemistry, vol. 283:3639-3654
(2008). cited by applicant .
Beiboer et al., "Guided selection of a pan carcinoma specific
antibody reveals similar binding characteristics yet structural
divergence between the original murine antibody and its human
equivalent," Journal of Molecular Biology, vol. 296:833-849 (2000).
cited by applicant .
Berglund et al., "Guided selection of a pan carcinoma specific
antibody reveals similar binding characteristics yet structural
divergence between the original murine antibody and its human
equivalent," Protein Science, vol. 17:606-613 (2008). cited by
applicant .
Berthelier et al., "Probing ligand-induced conformation changes of
human CD38," Eur. J. Biochem. 267:3056-64 (2000). cited by
applicant .
Borrione et al., "CD38 Stimulation Lowers the Activation Threshold
and Enhances the Alloreactivity of Cord Blood T Cells by Activating
the Phosphatidylinositol 3-Kinase Pathway and Inducing CD73
Expression," J. Immunology 162(10):6238-46 (1999). cited by
applicant .
Cesano et al., "Role of CD38 and its ligand in the regulation of
MHC-nonrestricted cytotoxic T cells," J. Immunology
160:1106-15(1998). cited by applicant .
Choi, Y. et al., "Predicting antibody complementarity determining
region structures without classification," Molecular BioSystems,
vol. 7:3327-3334 (2011). cited by applicant .
Corada, M. "Monoclonal antibodies directed to different regions of
vascular endothelial cadherin extracellular domain affect adhesion
and clustering of the protein and modulate endothelial
permeability," Blood, vol. 97:1679-84 (2001). cited by applicant
.
Corcoran, L. et al., "IL-5 and Rp105 Signaling Defects in B Cells
from Commonly Used 129 Mouse Substrains," The Journal of
Immunology, vol. 163(11):5836-5842 (1999). cited by applicant .
De Genst et al., "Antibody repertoire development in camelids,"
Developmental and Comparative Immunology, vol. 30:187-198 (2006).
cited by applicant .
European Search Report, European Application No. 19202827.2, dated
Jan. 28, 2020, 10 pages. cited by applicant .
Fedele et al., "CD38 is expressed on human mature monocyte-derived
dendritic cells and is functionally involved in CD83 expression and
IL-12 induction," Eur. J. Immunol. 34(5):1342-50 (2004). cited by
applicant .
Franco et al., "The transmembrane glycoprotein CD38 is a
Catalytically active transporter responsible for generation and
influx of the second messenger cyclic ADP-ribose across membranes,"
FASEB 12:1507-1520 (1998). cited by applicant .
Zupo et al., "Expression of CD5 and CD38 by human CD5-B cells:
requirement for special stimuli*" Eur. J. Immunology 24:1426-1433
(1994) ("Zupo 1994"). cited by applicant .
Giffilhs, A.D., et al., "Human anti-self antibodies with high
specificity from phage display libraries," The EMBO Journal, vol.
12:725-734(1993). cited by applicant .
Imanishi et al., "Post-Thymic Maturation of Migrating Human Thymic
Single-Positive T Cells: Thymic CD1a- CD4+ T Cells are More
Susceptible to Anergy Induction by Toxic Shock Syndrome Toxin-1
than Cord Blood CD4+ T Cells," J. Immunology 160(1)112-19 (1998).
cited by applicant .
Kitanaka et al., "CD38-Mediated Signaling Events in Murine Pro-B
Cells Expressing Human CD38 With or Without Its Cytoplasmic
Domain," J. Immunol. 162:1952-58 (1999). cited by applicant .
Klimka, A. et al., "Human anti-CD30 recombinant antibodies by
guided phage antibody selection using cell panning," British
Journal of Cancer, vol. 83:252-260 (2000). cited by applicant .
Korkut et al. "Serum proteins with NAD+ glycohydrolase activity and
anti-CD38 reactivity--elevated levels in serum of tumour patients,"
Cancer Letters 126:105-09 (1998). cited by applicant .
Kramer, G. et al., "High expression of a CD38-like molecule in
normal prostatic epithelium and its differential loss in benign and
malignant disease," The Journal of Urology, vol. 154(5):1636-1641
(1995). cited by applicant .
Kramer, G. et al., "Loss of CD38 correlates with simultaneous
up-regulation of human leukocyte antigen-DR in benign prostatic
glands, but not in fetal or androgen-ablated glands, and is
strongly related to gland atrophy," BJU International, vol. 91
(4):409-416 (2003). cited by applicant .
Kulkarni-Kale et al., "CEP: a conformational epitope prediction
server," Nucleic Acid Research, vol. 33:W168-W171 (2005). cited by
applicant .
Kumagai et al., "Ligation of CD38 Suppresses Human B
Lymphopoiesis," J. Exp. Med. 181:1101-10 (1995). cited by applicant
.
Malavasi et al., "Characterization of a Murine Monoclonal-Antibody
Specific for Human Early Lymphohemopoietic Cells," Hum Immunol
9:9-20 (1984). cited by applicant .
Mallone et al., "Anti-CD38 autoantibodies: characterisation in
new-onset type I diabetes and latent autoimmune diabetes of the
adult (LADA) and comparison with other islet autoantibodies,"
Diabetologia, 45(12):1667-1677 (2002). cited by applicant .
Mallone et al., "Autoantibody response to CD38 in Caucasian
patients with type 1 and type 2 diabetes immunological and genetic
characterization," Diabetes, 50(4):752-762 (2001). cited by
applicant .
Mallone et al., "Signaling through CD38 induces NK cell
activation," Int Immunol 13:397-409 (2001). cited by applicant
.
Mizuguchi et al., "Neuronal localization of CD38 antigen in the
human brain," Brain Research 697:235-240 (1995). cited by applicant
.
Morra et al., "CD38 is functionally dependent on the TCR/CD3
complex in human T cells," FASEB J. 12:581-92 (1998). cited by
applicant .
Padlan, E. et al., "X-ray crystallography of antibodies," Advances
in Protein Chemistry, vol. 49:57-133 (1996). cited by applicant
.
Pfister, M. et al., "NAD degradation and regulation of CD38
expression by human monocytes/macrophages," Eur. J. Biochem., vol.
268(21): 5601-5608 (2001). cited by applicant .
Presentation--Fully human antibodies for the treatment, 11th
Internal HAH conference, Oct. 6-8, 2004, Dublin MorphoSys HAH
Presentation, 33 pages (2004). cited by applicant .
Pupilli et al., "Autoantibodies to CD38 (ADP-ribosyl cyclase/cyclic
ADP-ribose hydrolase) in Caucasian patients with diabetes: effects
on insulin release from human islets," Diabetes, 48(12):2309-2315
(1999). cited by applicant .
Ruden, T. et al., "Fully human antibodies for the treatment of
severe diseases derived from the human combinatorial antibody
library HuCAL," Session 8: Molecular Biology-II on Oct. 7, 2004,
published in Human Antibodies 13 (2004), p. 27-30 Abstract Only.
cited by applicant .
Ruiz-Cabello et al., "A Monoclonal Antibody, GR7A4, Reacting with
the T10 Antigen," Hybridoma 6(3):275-84 (1987). cited by applicant
.
Sakalova et al., "Prognostic value of plasma-cell immunophenotype
in patients with multiple myeloma," Neoplasma, 40(6):351-354
(1993). cited by applicant .
Silvennoinen et al., "CD38 signal transduction in human B cell
precursors. Rapid induction of tyrosine phosphorylation, activation
of syk tyrosine kinase, and phosphorylation of phospholipase
C-gamma and phosphatidylinositol 3-kinase," J. Immunology
156:100-07 (1996). cited by applicant .
Sun et al., "A novel mechanism for coupling cellular intermediary
metabolism to cytosolic Ca2+ signaling via CD38/ADP-ribosyl
cyclase, a putative intracellular NAD+ sensor," FASEB 16(3):302-14
(2002). cited by applicant .
Tsujimoto, N. et al., "Potentiation of chemotactic peptide-induced
superoxide generation by CD38 ligation in human myeloid cell
lines," J. Biochem. 121(5): 949-956 (1997). cited by applicant
.
Ward, E. et al. "Binding activities of a repertoire of single
immunoglobulin variable domains secreted from Escherichia coli,"
Nature, vol. 341:544-546 (1989). cited by applicant .
Zilber et al., "CD38 expressed on human monocytes: A coaccessory
molecule in the superantigen-induced proliferation," PNAS
97(6):2840-45 (2000). cited by applicant .
Kropff, Martin H. et al., "Bortezomib in combination with
dexamethasone for relapsed multiple myeloma," Leukemia Research,
vol. 29:587-590 (2005). cited by applicant .
Lande, Roberto et al., "CD38 ligation plays a direct role in the
induction of IL-1b, IL-6, and IL-10 secretion in resting human
monocytes," Cellular Immunology, vol. 220:30-38 (2002). cited by
applicant .
Lazar, Eliane et al., "Transforming Growth Factor a: Mutation of
Aspartic Acid 47 and Leucine 48 Results in Different Biological
Activities," Molecular and Cellular Biology, vol. 8(3):1247-1252
(1988). cited by applicant .
Lin, Michael C. et al., "Structure-Function Relationships in
Glucagon: Properties of Highly Purified Des-His-, Monoiodo-, and
[Des-Asn28, Thr29](homoserine Iactone27)-glucagon," Biochemistry,
vol. 14(8):1559-1563 (1975). cited by applicant .
Lonberg, Nils et al., "Antigen-specific human antibodies from mice
comprising four distinct genetic modifications," Nature, vol.
368:856-859 (1994). cited by applicant .
MacCallum, Robert M. et al., "Antibody-antigen Interactions:
Contact Analysis and Binding Site Topography," J. Mol. Biol., vol.
262:732-745 (1996). cited by applicant .
Malavasi, Fabio et al., "Human CD38: a glycoprotein in search of a
function," Immunology Today, vol. 15(3):95-97 (1994). cited by
applicant .
Maloney, David G. et al., "Antibody Therapy for Treatment of
Multiple Myeloma," Seminars in Hematology, vol. 36(1 Suppl.
3):30-33 (1999). cited by applicant .
Mills, Charity et al., "Characterization of Monoclonal Antibodies
that Inhibit CD38 ADP-Ribosyl Cyclase Activity," Poster with
abstract presented at a student conference at the University of
Minnesota (2007). cited by applicant .
Mukherjee, Jean et al., "Production and Characterization of
Protective Human Antibodies against Shiga Toxin 1," Infection and
Immunity, vol. 70(10):5896-5899 (2002). cited by applicant .
NCBI NP.sub.--001766 for human CD38, lasted updated Jun. 1, 2014.
cited by applicant .
Orlowski, Robert Z., "The Ubiquitin Proteasome Pathway from Bench
to Bedside," American Society of Hematology, pp. 220-225 (2005).
cited by applicant .
Osterborg, Anders et al., "Natural Interferon-alpha in Combination
With Melphalan/Prednisone Versus Melphalan/Prednisone in the
Treatment of Multiple Myeloma Stages II and III: A Randomized Study
From the Myeloma Group of Central Sweden," Blood, vol.
81(6):1428-1434 (1993). cited by applicant .
Padlan, Eduardo A. et al., "Identification of
specificity-determining residues in antibodies," Faseb J., vol.
9:133-139 (1995). cited by applicant .
Parren, "HuMax-CD38," Conference Proceeding, Presentation for the
23rd International Conference on Advances in the Application of
Monoclonal Antibodies in Clinical Oncology, Myconos, Greece (2006).
cited by applicant .
Parren, "HuMax-CD38," Conference Proceedings, Presentation for the
CD38 metting in Torino (2006). cited by applicant .
Parren, P.W.H.I. et al., "HuMax-CD38, a new human CD38 monoclonal
antibody, effectively mediates killing of multiple myeloma and
plasma cell leukemia cells," PWHI Conference Proceeding,
Presentation for the CD38 meeting in Torino, Jun. 8-10, 2006. cited
by applicant .
Paul, William E., "Fv Structure and Diversity in Three Dimensions,"
Fundamantal Immunology, Third Edition, Raven Press, New York, pp.
292-295 (1993). cited by applicant .
Peipp, M. et al., "Fully Human CD38 Antibodies Efficiently Trigger
ADCC and CDC of Multiple Myeloma and Plasma Cell Leukemia Cells,"
Conference Proceedings, Poster Presentation at the 2005 Annual
Meeting of the American Society of Hematology, 1 page, Dec. 12,
2005. cited by applicant .
Peipp, Matthias et al., "Fully Human CD38 Antibodies Efficiently
Trigger ADCC and CDC of Multiple Myeloma Cell Lines and Primary
Tumor Cells," Conference Proceedings, Poster presentation of the
2005 Annual Meeting of the American Society of Hematology, 1 page
(2005). cited by applicant .
Peipp, Matthias et al., AN PREV200600185745, "Fully human CD38
antibodies efficiently trigger ADCC of multiple myeloma cell lines
and primary tumor cells," Blood, vol. 106(11):944A, 47th Annual
Meeting of the American-Society-of-Hematology (2005). cited by
applicant .
Peng, Kah-Whye et al., "Oncolytic measles viruses displaying a
single-chain antibody against CD38, a myeloma cell marker," Blood,
vol. 101(7):2557-2562 (2003). cited by applicant .
PJ Carter, Nat Rev Immunol, 2006; 6:343-357. cited by applicant
.
Rudikoff, Stuart et al., "Single amino acid substitution altering
antigen-binding specificity," Proc. Natl. Acad. Sci., USA, vol.
79:1979-1983 (1982). cited by applicant .
Schwartz, Gerald P., "A superactive insulin: [B10-Aspartic
acid]insulin(human)," Proc. Natl. Acad. Sci. USA, vol. 84:6408-6411
(1987). cited by applicant .
Shimazaki, Chihiro, "Advances in the Treatment of Multiple
Myeloma--standard early-stage treatment," Medical Practice, vol.
22(8): 1395-1398 (2005). cited by applicant .
Shubinsky & Schlesinger, Immunity 1997; 7:315-24. cited by
applicant .
Skolnick, Jeffrey et al., "From genes to protein structure and
function: novel applications of computational approaches in the
genomic era," TibTech, vol. 18:34-39 (2000). cited by applicant
.
Stevenson, Freda K. et al., "Preliminary Studies for an
Immunotherapeutic Approach to the Treatment of Human Myeloma Using
Chimeric Anti-CD38 Antibody," Blood, vol. 77(5):1071-1079 (1991).
cited by applicant .
Stevenson, George T., "CD38 as a Therapeutic Target," Mol. Med.,
vol. 12(11-12):345-346 (2006). cited by applicant .
Takasawa, Shin et al., "Synthesis and Hydrolysis of Cyclic
ADP-Ribose by Human Leukocyte Antigen CD38 and Inhibition of the
Hydrolysis by ATP," The Journal of Biological Chemistry, vol.
268(35):26052-26054 (1993). cited by applicant .
Tamura, Midori et al., "Structural Correlates of an Anticarcinoma
Antibody: Identification of Specificity-Determining Residues (SDRs)
and Development of a Minimally Immunogenic Antibody Variant by
Retention of SDRs Only," The Journal of Immunology, vol.
164:1432-1441 (2000). cited by applicant .
Terada, Hideo, "What is multiple myeloma?" Modern Physician, vol.
26(5):883-887 (2006). cited by applicant .
Vajdos, Felix F. et al., "Comprehensive Functional Maps of the
Antigen-binding Site of an Anti-ErbB2 Antibody Obtained with
Shotgun Scanning Mutagenesis," J. Mol. Biol., vol. 320:415-428
(2002). cited by applicant .
Van Der Veer, Michael S. et al., "Towards effective immunotherapy
of myeloma: enhanced elimination of myeloma cells by combination of
lenalidomide with the human CD38 monoclonal antibody daratumumab,"
Haematologica, vol. 96(2):284-290 (2011). cited by applicant .
Van Spriel, Annemiek B. et al., "Immunotherapeutic perspective for
bispecific antibodies," Immunology Today, vol. 21(8):391-397
(2000). cited by applicant .
Vooijs, W.C. et al., "Evaluation of CD38 as Target for
Immunotherapy in Multiple Myeloma," Blood, vol. 85 (8):2282-2284
(1995). cited by applicant .
Wiesenthal, "Synergy analysis of `classic` and newer drug
combinations," Human Tumor Assay Journal, retrieved online at:
http://weisenthal.org/synergy1.htm, 1 page (2012). cited by
applicant .
Written Opinion for Application No. PCT/DK2006/000166, dated Sep.
25, 2007. cited by applicant .
Wu, Herren et al., "Humanization of a Murine Monoclonal Antibody by
Simultaneous Optimization of Framework and CDR Residues," J. Mol.
Biol., vol. 294:151-162 (1999). cited by applicant .
Yamashita, Y. et al., "A monoclonal antibody against a murine CD38
homologue delivers a signal to B cells for prolongation of survival
and protection against apoptosis in vitro: unresponsiveness of
X-linked immunodeficient B cells," Immunology, vol. 85:248-255
(1995). cited by applicant .
Zocchi, Elena et al., "A Single Protein Immunologically Identified
as CD38 Displays NAD+ Glycohydrolase, ADP-Ribosyl Cyclase and
Cyclic ADP-Ribose Hydrolase Activities at the Outer Surface of
Human Erythrocytes," Biochemical and Biophysical Research
Communications, vol. 196(3):1459-1465 (1993). cited by applicant
.
Zubiaur, Mercedes et al., "CD38 Ligation Results in Activation of
the Raf-1/Mitogen-Activated Protein Kinase and the
CD3-z/z-Associated Protein-70 Signaling Pathways in Jurkat T
Lymphocytes," The Journal of Immunology, vol. 159:193-205 (1997).
cited by applicant .
Aarhus, Robert et al., "ADP-ribusyl Cyclase and CD38 Catalyze the
Synthesis of a Calcium-mobilizing Metabolite from NADP," The
Journal of Biological Chemistry, vol. 270(51):30327-30333 (1995).
cited by applicant .
Abebanjo, Olugbenga A. et al., "A new function for CD38/ADP-ribosyl
cyclase in nuclear ca2+ homeostasis," Nature Cell Biology, vol.
1:409-414 (1999). cited by applicant .
Adams, Julian et al., "Proteasome inhibition: a new strategy in
cancer treatment," Investigational New Drugs, vol. 18:109-121
(2000). cited by applicant .
Almagro & Fransson, Frontiers in Bioscience 2008; 13:1619-33.
cited by applicant .
Antonelli, Alessandro et al., "Human Anti-CD38 Autoantibodies Raise
Intracellular Calcium and Stimulate Insulin Release in Human
Pancreatic Islets," Diabetes, vol. 50:985-991 (2001). cited by
applicant .
ASH 2014 Abstract #3474, Direct in Vitro Comparison of Daratumumab
with Surrogate Analogs of CD38 Antibodies MOR03087, SAR650984 and
Ab79, Lammerts van Bueren et al., Oral and Poster Abstracts, Dec.
7, 2014. cited by applicant .
Ausiello, C.M. et al., "Functional topography of discrete domains
of human CD38," Tissue Antigens, vol. 56:539-547 (2000). cited by
applicant .
Berenbaum, M.C., "Synergy, additivism and antagonism in
immunosuppression, A Critical Review," Clin. Exp. Immunol., vol.
28:1-18(1977). cited by applicant .
Boccadoro, Mario et al., "Preclinical evaluation of the proteasome
inhibitor bortezomib in cancer therapy," Cancer Cell International,
vol. 5(18):1-9 doi:10.1186/1475-2867-5-18 (2005). cited by
applicant .
Bolognesi, A. et al., "CD38 as a target of IB4 mAb carrying
saporin-S6: Design of an immunotoxin for ex vivo depletion of
hematological CD38+ neoplasia," Journal of Biological Regulators
and Homeostatic Agents, vol. 19:145-152 (2005). cited by applicant
.
Burgess, Wilson H. et al., "Possible Dissociation of the
Heparin-binidng and Mitogenic Activities of Heparin-binding (Acidic
Fibroblast Growth Factor-1 from Its Receptor-binding Activities by
Site-directed Mutagenesis of a Single Lysine Residue," The Journal
of Cell Biology, vol. 111:2129-2138 (1990). cited by applicant
.
Casset, Florence et al., "A peptide mimetic of an anti-CD4
monoclonal antibody by rational design," Biochemical and
Biophysical Research Communications, vol. 307:198-205 (2003). cited
by applicant .
Cavo, Michele et al., "Superiority of thalidomide and dexamethasone
over vincristine-doxorubicin-dexamethasone (VAD) as primary therapy
in preparation for autologous transplantation for multiple
myeloma," Blood, vol. 106(1):35-39 (2005). cited by applicant .
Chen, Yvonne et al., "Selection and Analysis of an Optimized
Anti-VEGF Antibody: Crystal Structure of an Affinity-matured Fab in
Complex with Antigen," J. Mol. Biol., vol. 293:865-881 (1999).
cited by applicant .
Chou, Ting-Chao, "Drug Combination Studies and Their Synergy
Quantification Using the Chau-Talalay Method," Cancer Res., vol.
70(2):440-446 (2010). cited by applicant .
Colman, P.M., "Effects of amino acid sequence changes on
anibody-antigen interactions," Research in Immunology, vol.
145:33-36(1994). cited by applicant .
Cotner, Thomas et al., "Human T Cell Proteins Recognized by Rabbit
Heteroantisera and Monoclonal Antibodies," Int. J. Immunopharmac.,
vol. 3(3):255-268 (1981). cited by applicant .
Davies, Julian et al., "Affinity improvement of single antibody VH
domains: residues in all three hypervariable regions affect antigen
binding," Immunotechnology, vol. 2:169-179 (1996). cited by
applicant .
De Weers, M. et al., "Humax-CD38, a New Human CD38 Monoclonal
Antibody, Effectively Mediates Killing of Multiple Myeloma and
Plasma Cell Leukemia Cells," abstract, submitted for the 16th
European Congress of Immunology--ECI2006. Sep. 6-9, 2006--Paris,
France. cited by applicant .
De Weers, M. et al., "HuMax-CD38, a new human CD38 monoclonal
antibody, effectively mediates killing of multiple myeloma and
plasma cell leukemia cells," Genmab, 1 page (2006). cited by
applicant .
De Weers, M. et al., "HuMax-CD38, a new human CD38 monoclonal
antibody, effectively mediates killing of multiple myeloma and
plasma cell leukemia cells," Poster presented at the 1st Joint
Meeting of European National Societies of Immunology under auspices
of EFIS, Sep. 6-9, 2006. cited by applicant .
De Weers, Michel et al., "Daratumumab, a Novel Therapeutic Human
CD38 Monoclonal Antibody, Induces Killing of Multiple Myeloma and
Other Hematological Tumors," The Journal of Immunology, vol.
186:1840-1848 (2011). cited by applicant .
De Weers, Michel, "HuMax-CD38," Presentation at the Regional
Myeloma Group Meeting (2007). cited by applicant .
Deckert et al., Clin. Cancer Res. 2014; 20:4574-83. cited by
applicant .
Donovan, K.A. et al., "Binding and internalization of an antibody
engineered ant-CD38 single chain variable fragment KscFv) by human
myeloma cells," Blood, vol. 90(10):88A (1997). cited by applicant
.
Ellis, Jonathan H. et al., "Engineered Anti-CD38 Monoclonal
Antibodies for Immunotherapy of Multiple Myeloma," The Journal of
Immunology, vol. 155:925-937 (1995). cited by applicant .
Ferrero, Enza et al., "Characterization and phylogenetic epitope
mapping of CD38 ADPR cyclase in the cynomolgus macaque," BMC
Immunology, vol. 5(21):1-13 doi10.1186/1471-2172-5-21 (2004). cited
by applicant .
Field-Smith, Antonia et al., "Bortezomib (Velcade) in the treatment
of multiple myeloma," Therapeutics and Clinical Risk Management,
vol. 2(3):271-279 (2006). cited by applicant .
Funaro, Ada et al., "CD38 Functions Are Regulated Through an
Internalization Step," The Journal of Immunology, vol.
160:2238-2247 (1998). cited by applicant .
Funaro, Ada et al., "Human CD38: a versatile leukocyte molecule
with emerging clinical perspectives," Fundamental and Clinical
Immunology, vol. 3(3):101-113 (1995). cited by applicant .
Funaro, Ada et al., "Identification and characterization of an
active soluble form of human CD38 in normal and pathological
fluids," International Immunology, vol. 8(11):1643-1650 (1996).
cited by applicant .
Funaro, Ada et al., "Involvement of the multilineage CD38 molecule
in a unique pathway of cell activation and proliferation," The
Journal of Immunology, vol. 145(8):2390-2396 (1990). cited by
applicant .
Genmab Post-ASH Seminar, Dec. 2013, pp. 1 and 70-73. cited by
applicant .
Genmab, "Humax-CD38 Effective in Preclinical Studies," retrieved
online at
/findarticles.com/p/articles/mi.sub.--hb5570/is.sub.--200512/ai.sub.--n24-
- 200986 (2005). cited by applicant .
Goldmacher, Victor S. et al., "Anti-CD38-Blocked Ricin: An
Immunotoxin for the Treatment of Multiple Myeloma," Blood, vol.
84(9):3017-3025 (1994). cited by applicant .
Graeff, Richard M. et al., "Enzymatic Synthesis and
Characterizations of Cyclic GDP-ribose. A Procedure for
Distinguishing Enzymes with ADP-Ribosyl Cyclase Activity," The
Journal of Biological Chemistry, vol. 269 (48):30260-30267 (1994).
cited by applicant .
Green, L.L. et al., "Antigen-specific human monoclonal antibodies
from mice engineered with human Ig heavy and light chain YACs,"
Nature Genetics, vol. 7:13-21 (1994). cited by applicant .
Hara-Yokoyama, Miki et al., "Alteration of enzymatic properties of
cell-surface antigen CD38 by agonistic anti-CD38 antibodies that
prolong B cell survival and induce activation," International
Immunopharmacology, vol. 8:59-70 (2008). cited by applicant .
Holm, Patrik et al., "Functional mapping and single chain
construction of the anti-cytokeratin 8 monoclonal antibody TS1,"
Molecular Immunology, vol. 44:1075-1084 (2007). cited by applicant
.
Holt, Lucy J. et al., "Domain antibodies: proteins for therapy,"
Trends in Biotechnology, vol. 21(11):484-490 (2003). cited by
applicant .
Hoshino, Shin-ichi et al., "Mapping of the Catalytic and Epitopic
Sites of Human CD38/NAD+ Glycohydrolase to a Functional Domain in
the Carboxyl Terminus," The Journal of Immunology, vol. 158:741-747
(1997). cited by applicant .
Howard, Maureen et al., "Formation and Hydrolysis of Cyclic
ADP-Ribose Catalyzed by Lymphocyte Antigen CD38," Science, vol.
262:1056-1059 (1993). cited by applicant .
Ikehata, Fumiko et al., "Autoantibodies against CD38 (ADP-ribosyl
Cyclase/Cyclic ADP-ribose Hydrolase) that Impair Glucose-induced
Insulin Secretion in Noninsulin-dependent Diabetes Patients," J.
Clin. Invest., vol. 102(2):395-401 (1998). cited by applicant .
International Search Report for Application No. PCT/DK2006/000166,
dated Aug. 14, 2006. cited by applicant .
International Search Report for Application No. PCT/EP2011/059507,
6 pages, dated Sep. 6, 2011. cited by applicant .
Jackson, David G. et al., "Isolation of a cDNA Encoding the Human
CD38 (T10) Molecule, a Cell Surface Glycoprotein with an Unusual
Discontinuous Pattern of Expression During Lymphocyte
Differentiation," The Journal of Immunology, vol. 144(7):2811-2815
(1990). cited by applicant .
Jagannath, Sundar, "Multiple Myeloma Update from the American
Society of Clinical Oncology (ASCO) 41st Annual Meeting," Update
from the American Society of Clinical Oncology (ASCO) 41st Annual
Meeting: Poster Sessions, 3 pages (2005). cited by applicant .
Johnson, Malisha R. et al., "Primary plasma cell leukemia:
morphologic immunophenotypic, and cytogenetic featues of 4 cases
treated with chemotherapy and stem cell transplantation," Annals of
Diagnostic Pathology, vol. 10:263-268 (2006). cited by applicant
.
Konopleva, Marina et al., "CD38 in Hematopoietic Malignancies,"
Human CD38 and Related Molecules.Chem Immunol., vol. 75:189-206
(2000). cited by applicant .
Konopleva, Marina et al., "Ligation of Cell Surface CD38 Protein
with Agonistic Monoclonal Antibody Induces a Cell Growth Signal in
Myeloid Leukemia Cells," The Journal of Immunology, vol.
161:4702-4708 (1998). cited by applicant .
Peng, K-W. et al., "Oncolytic measles viruses displaying a
single-chain antibody against CD38, a myeloma cell marker," Blood,
vol. 101(7) 2557-2562 (2003). cited by applicant .
Davis et al. "Production of human antibodies from transgenic mice,"
Methods in Molecular Biology, Antibody Engineering, vol. 248, 14
pages (2004). cited by applicant.
|
Primary Examiner: Roark; Jessica H
Attorney, Agent or Firm: Nelson Mullins Riley &
Scarborough LLP Remillard, Esq.; Jane E. Frank; Christopher L.
Parent Case Text
RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser.
No. 14/990,869, filed Jan. 8, 2016 (now U.S. Pat. No. 9,944,711),
which is a divisional of U.S. patent application Ser. No.
13/702,857, filed Dec. 17, 2012 (now U.S. Pat. No. 9,249,226),
which is a 35 U.S.C. 371 national stage filing of International
Application No. PCT/EP2011/059507, filed Jun. 8, 2011.
International Application No. PCT/EP2011/059507 claims priority to
U.S. Provisional Application No. 61/353,082, filed Jun. 9, 2010,
and Danish Patent Application No. PA 2010 00498, filed Jun. 9,
2010.
Claims
The invention claimed is:
1. A nucleic acid encoding an antibody that binds to human CD38 as
set forth in SEQ ID NO: 52, wherein the antibody comprises: (i) a
VH CDR1 having the sequence as set forth in any of the sequences
SEQ ID NOs: 3, 8, 13, 18, and 23, a VH CDR2 having the sequence as
set forth in any of the sequences SEQ ID NOs: 4, 9, 14, 19, and 24,
a VH CDR3 having the sequence as set forth in any of the sequences
SEQ ID NOs: 5, 10, 15, 20, and 25, a VL CDR1 having the sequence as
set forth in any of the sequences SEQ ID NOs: 28, 33, 38, 43, and
48, a VL CDR2 having the sequence as set forth in any of the
sequences SEQ ID NOs: 29, 34, 39, 44, and 49, and a VL CDR3 having
the sequence as set forth in any of the sequences SEQ ID NOs: 30,
35, 40, 45, and 50, (ii) a VH CDR1 having the sequence as set forth
in SEQ ID NO: 3, a VH CDR2 having the sequence as set forth in SEQ
ID NO: 4, a VH CDR3 having the sequence as set forth in SEQ ID NO:
5, a VL CDR1 having the sequence as set forth in SEQ ID NO: 28, a
VL CDR2 having the sequence as set forth in SEQ ID NO: 29, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 30, (iii) a VH
CDR1 having the sequence as set forth in SEQ ID NO: 8, a VH CDR2
having the sequence as set forth in SEQ ID NO: 9, a VH CDR3 having
the sequence as set forth in SEQ ID NO: 10, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 33, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 34, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 35, (iv) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 13, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 14, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 15, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 38, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 39, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 40, (v) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 18, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 19, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 20, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 43, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 44, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 45, or (vi) a VH CDR1 having
the sequence as set forth in SEQ ID NO: 23, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 24, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 25, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 48, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 49, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 50.
2. The nucleic acid of claim 1, wherein the antibody does not bind
to a variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human
CD38.
3. The nucleic acid of claim 2, wherein the EC50 of the binding of
the antibody to the variant of human CD38 wherein Asp in position
202 has been substituted with Gly is less than 50%.
4. The nucleic acid of claim 1, wherein the antibody binds to a
variant of human CD38 wherein Gln in position 272 has been
substituted with Arg to the same degree that it binds to human
CD38.
5. The nucleic acid of claim 4, wherein the EC50 of the binding of
the antibody to the variant of human CD38 wherein Gln in position
272 has been substituted with Arg is at least 80% of the EC50 of
the binding of the antibody to human CD38.
6. The nucleic acid of claim 1, wherein the antibody binds to a
variant of human CD38 wherein the Ser in position 274 has been
substituted with Phe to the same degree that it binds to human
CD38.
7. The nucleic acid of claim 6, wherein the EC50 of the binding of
the antibody to the variant of human CD38 is at least 75% of the
EC50 of the binding of the antibody to human CD38.
8. The nucleic acid of claim 1, wherein the antibody possesses the
following binding characteristics: (i) it does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human
CD38, (ii) it binds to a variant of human CD38 wherein Gln in
position 272 has been substituted with Arg to the same degree that
it binds to human CD38, and (iii) it binds to a variant of human
CD38 wherein the Ser in position 274 has been substituted with Phe
to the same degree that it binds to human CD38.
9. The nucleic acid of claim 1, wherein the antibody binds human
CD38 and has an inhibitory effect on CD38 cyclase activity and a
stimulatory effect on CD38 hydrolase activity.
10. The nucleic acid of claim 9, wherein the inhibitory effect is
at least 50-66% compared to the inhibitory effect on the CD38
cyclase activity in the absence of the antibody.
11. The nucleic acid of claim 1, wherein the antibody is capable of
inducing antibody-dependent cellular cytotoxicity (ADCC).
12. The nucleic acid of claim 1, wherein the antibody is not
capable of inducing ADCC in Daudi cells.
13. The nucleic acid of claim 1, wherein the antibody is not
capable of inducing complement-dependent cytotoxicity (CDC) in
CHO-CD38 cells.
14. The nucleic acid of claim 1, wherein the antibody binds to
human CD38 with a K.sub.D of 10.sup.-8 M or less.
15. The nucleic acid of claim 1, wherein the antibody is a human
monovalent antibody.
16. The nucleic acid of claim 1, wherein the antibody is a full
length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody.
17. The nucleic acid of claim 1, wherein the antibody is an
antibody fragment or a single-chain antibody.
18. The nucleic acid of claim 1, wherein the antibody is an
effector-function-deficient antibody.
19. The nucleic acid of claim 1, wherein the antibody is a
monovalent antibody.
20. The nucleic acid of claim 1, wherein the antibody inhibits the
CD38 catalyzed synthesis of cGDPR by at least 25%.
21. The nucleic acid of claim 1, wherein the antibody inhibits the
CD38 catalyzed synthesis of cADPR by at least 25%.
22. The nucleic acid of claim 1, wherein the antibody stimulates
the hydrolase activity of CD38 by at least 25%.
23. The nucleic acid of claim 1, wherein the antibody stimulates
the NAD hydrolase activity of CD38 by at least 25%.
24. The nucleic acid of claim 1, wherein the antibody stimulates
the cADPR-hydrolase activity of CD38 by at least 25%.
25. The nucleic acid of claim 1, wherein the antibody inhibits the
ability of CD38 to catalyze the formation, via a base-exchange
reaction, of NAADP with an IC50 of below 0.5 .mu.g/mL.
26. The nucleic acid of claim 1, wherein the antibody is a
bispecific antibody that has a second binding specificity for a
human effector cell or a cancer antigen.
27. An expression vector comprising the nucleic acid of claim
1.
28. A recombinant eukaryotic or prokaryotic host cell which
comprises the expression vector of claim 27.
29. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 28, and b) purifying the anti-CD38 antibody from the
culture media.
30. A recombinant eukaryotic or prokaryotic host cell which
comprises the nucleic acid of claim 1.
31. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 30, and b) purifying the anti-CD38 antibody from the
culture media.
32. The nucleic acid of claim 1, wherein the antibody comprises a
VH CDR1 having the sequence as set forth in SEQ ID NO: 3, a VH CDR2
having the sequence as set forth in SEQ ID NO: 4, a VH CDR3 having
the sequence as set forth in SEQ ID NO: 5, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 28, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 29, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 30.
33. A recombinant eukaryotic or prokaryotic host cell which
comprises an expression vector comprising the nucleic acid of claim
32.
34. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 33, and b) purifying the anti-CD38 antibody from the
culture media.
35. An expression vector comprising the nucleic acid of claim
32.
36. A recombinant eukaryotic or prokaryotic host cell which
comprises the expression vector of claim 35.
37. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 36, and b) purifying the anti-CD38 antibody from the
culture media.
38. A recombinant eukaryotic or prokaryotic host cell which
comprises the nucleic acid of claim 32.
39. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 38, and b) purifying the anti-CD38 antibody from the
culture media.
40. The nucleic acid of claim 1, wherein the antibody comprises a
VH CDR1 having the sequence as set forth in SEQ ID NO: 8, a VH CDR2
having the sequence as set forth in SEQ ID NO: 9, a VH CDR3 having
the sequence as set forth in SEQ ID NO: 10, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 33, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 34, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 35.
41. The nucleic acid of claim 1, wherein the antibody comprises a
VH CDR1 having the sequence as set forth in SEQ ID NO: 13, a VH
CDR2 having the sequence as set forth in SEQ ID NO: 14, a VH CDR3
having the sequence as set forth in SEQ ID NO: 15, a VL CDR1 having
the sequence as set forth in SEQ ID NO: 38, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 39, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 40.
42. The nucleic acid of claim 1, wherein the antibody comprises a
VH CDR1 having the sequence as set forth in SEQ ID NO: 18, a VH
CDR2 having the sequence as set forth in SEQ ID NO: 19, a VH CDR3
having the sequence as set forth in SEQ ID NO: 20, a VL CDR1 having
the sequence as set forth in SEQ ID NO: 43, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 44, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 45.
43. The nucleic acid of claim 1, wherein the antibody comprises a
VH CDR1 having the sequence as set forth in SEQ ID NO: 23, a VH
CDR2 having the sequence as set forth in SEQ ID NO: 24, a VH CDR3
having the sequence as set forth in SEQ ID NO: 25, a VL CDR1 having
the sequence as set forth in SEQ ID NO: 48, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 49, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 50.
44. A nucleic acid comprising one or more nucleotide sequences
selected from the group consisting of SEQ ID NOs: 1, 6, 11, 16, 21,
26, 31, 36, 41, and 46.
45. The nucleic acid of claim 44, which comprises the nucleotide
sequences set forth in SEQ ID NOs: 1 and 26, SEQ ID NOs: 6 and 31,
SEQ ID NOs: 11 and 36, SEQ ID NOs: 16 and 41, or SEQ ID NOs: 21 and
46.
46. A nucleic acid encoding an antibody that binds to CD38, wherein
the antibody comprises a VH region comprising any one of the
sequences as set forth in SEQ ID NOs: 2, 7, 12, 17, and 22, and a
VL region comprising any one of the sequences as set forth in SEQ
ID NOs: 27, 32, 37, 42, and 47.
47. The nucleic acid of claim 46, wherein the antibody comprises:
(i) a VH region comprising the sequence as set forth in SEQ ID NO:
2, and a VL region comprising the sequence as set forth in SEQ ID
NO: 27, (ii) a VH region comprising the sequence as set forth in
SEQ ID NO: 7, and a VL region comprising the sequence as set forth
in SEQ ID NO: 32, (iii) a VH region comprising the sequence as set
forth in SEQ ID NO: 12, and a VL region comprising the sequence as
set forth in SEQ ID NO: 37, (iv) a VH region comprising the
sequence as set forth in SEQ ID NO: 17, and a VL region comprising
the sequence as set forth in SEQ ID NO: 42, or (v) a VH region
comprising the sequence as set forth in SEQ ID NO: 22, and a VL
region comprising the sequence as set forth in SEQ ID NO: 47.
48. The nucleic acid of claim 47, wherein the antibody comprises a
VH region comprising the sequence as set forth in SEQ ID NO: 2, and
a VL region comprising the sequence as set forth in SEQ ID NO:
27.
49. A recombinant eukaryotic or prokaryotic host cell which
comprises an expression vector comprising the nucleic acid of claim
48.
50. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 49, and b) purifying the anti-CD38 antibody from the
culture media.
51. An expression vector comprising the nucleic acid of claim
48.
52. A recombinant eukaryotic or prokaryotic host cell which
comprises the expression vector of claim 51.
53. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 52, and b) purifying the anti-CD38 antibody from the
culture media.
54. A recombinant eukaryotic or prokaryotic host cell which
comprises the nucleic acid of claim 48.
55. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 54, and b) purifying the anti-CD38 antibody from the
culture media.
56. The nucleic acid of claim 47, wherein the antibody comprises a
VH region comprising the sequence as set forth in SEQ ID NO: 7, and
a VL region comprising the sequence as set forth in SEQ ID NO:
32.
57. The nucleic acid of claim 47, wherein the antibody comprises a
VH region comprising the sequence as set forth in SEQ ID NO: 12,
and a VL region comprising the sequence as set forth in SEQ ID NO:
37.
58. The nucleic acid of claim 47, wherein the antibody comprises a
VH region comprising the sequence as set forth in SEQ ID NO: 17,
and a VL region comprising the sequence as set forth in SEQ ID NO:
42.
59. The nucleic acid of claim 47, wherein the antibody comprises a
VH region comprising the sequence as set forth in SEQ ID NO: 22,
and a VL region comprising the sequence as set forth in SEQ ID NO:
47.
60. The nucleic acid of claim of claim 47, wherein the antibody is
capable of inducing antibody-dependent cellular cytotoxicity
(ADCC).
61. The nucleic acid of claim of claim 47, wherein the antibody is
not capable of inducing ADCC in Daudi cells.
62. The nucleic acid of claim 47, wherein the antibody is not
capable of inducing complement-dependent cytotoxicity (CDC) in
CHO-CD38 cells.
63. The nucleic acid of claim 47, wherein the antibody binds to
human CD38 with a K.sub.D of 10.sup.-8 M or less.
64. The nucleic acid of claim 47, wherein the antibody is a human
monovalent antibody.
65. The nucleic acid of claim 47, wherein the antibody is a full
length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody.
66. The nucleic acid of claim 47, wherein the antibody is an
antibody fragment or a single-chain antibody.
67. The nucleic acid of claim 47, wherein the antibody is an
effector-function-deficient antibody.
68. The nucleic acid of claim 47, wherein the antibody is a
monovalent antibody.
69. The nucleic acid of claim 47, wherein the antibody is a
bispecific antibody that has a second binding specificity for a
human effector cell or a cancer antigen.
70. An expression vector comprising the nucleic acid of claim
47.
71. A recombinant eukaryotic or prokaryotic host cell which
comprises the expression vector of claim 70.
72. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 71, and b) purifying the anti-CD38 antibody from the
culture media.
73. A recombinant eukaryotic or prokaryotic host cell which
comprises the nucleic acid of claim 47.
74. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, wherein the antibody does
not bind to a variant of human CD38 wherein Asp in position 202 has
been substituted with Gly to the same degree that it binds to human
CD38, the method comprising the steps of: a) culturing the host
cell of claim 73, and b) purifying the anti-CD38 antibody from the
culture media.
75. A combination of nucleic acids encoding an antibody that binds
to CD38, wherein the antibody comprises: (i) a VH CDR1 having the
sequence as set forth in any of the sequences SEQ ID NOs: 3, 8, 13,
18, and 23, a VH CDR2 having the sequence as set forth in any of
the sequences SEQ ID NOs: 4, 9, 14, 19, and 24, a VH CDR3 having
the sequence as set forth in any of the sequences SEQ ID NOs: 5,
10, 15, 20, and 25, a VL CDR1 having the sequence as set forth in
any of the sequences SEQ ID NOs: 28, 33, 38, 43, and 48, a VL CDR2
having the sequence as set forth in any of the sequences SEQ ID
NOs: 29, 34, 39, 44, and 49, and a VL CDR3 having the sequence as
set forth in any of the sequences SEQ ID NOs: 30, 35, 40, 45, and
50, (ii) a VH CDR1 having the sequence as set forth in SEQ ID NO:
3, a VH CDR2 having the sequence as set forth in SEQ ID NO: 4, a VH
CDR3 having the sequence as set forth in SEQ ID NO: 5, a VL CDR1
having the sequence as set forth in SEQ ID NO: 28, a VL CDR2 having
the sequence as set forth in SEQ ID NO: 29, and a VL CDR3 having
the sequence as set forth in SEQ ID NO: 30, (iii) a VH CDR1 having
the sequence as set forth in SEQ ID NO: 8, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 9, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 10, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 33, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 34, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 35, (iv) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 13, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 14, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 15, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 38, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 39, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 40, (v) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 18, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 19, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 20, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 43, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 44, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 45, or (vi) a VH CDR1 having
the sequence as set forth in SEQ ID NO: 23, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 24, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 25, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 48, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 49, and a VL CDR3 having the
sequence as set forth in SEQ ID NO: 50.
76. The combination of nucleic acids of claim 75, wherein the
antibody comprises a VH CDR1 having the sequence as set forth in
SEQ ID NO: 3, a VH CDR2 having the sequence as set forth in SEQ ID
NO: 4, a VH CDR3 having the sequence as set forth in SEQ ID NO: 5,
a VL CDR1 having the sequence as set forth in SEQ ID NO: 28, a VL
CDR2 having the sequence as set forth in SEQ ID NO: 29, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 30.
77. A combination of expression vectors comprising the combination
of nucleic acids of claim 76.
78. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of expression vectors of claim 77.
79. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 78, and b) purifying
the anti-CD38 antibody from the culture media.
80. An expression vector comprising the combination of nucleic
acids of claim 76.
81. A recombinant eukaryotic or prokaryotic host cell comprising
the expression vector of claim 80.
82. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 81, and b) purifying
the anti-CD38 antibody from the culture media.
83. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of nucleic acids of claim 76.
84. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 83, and b) purifying
the anti-CD38 antibody from the culture media.
85. The combination of nucleic acids of claim 75, wherein the
antibody comprises a VH CDR1 having the sequence as set forth in
SEQ ID NO: 8, a VH CDR2 having the sequence as set forth in SEQ ID
NO: 9, a VH CDR3 having the sequence as set forth in SEQ ID NO: 10,
a VL CDR1 having the sequence as set forth in SEQ ID NO: 33, a VL
CDR2 having the sequence as set forth in SEQ ID NO: 34, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 35.
86. The combination of nucleic acids of claim 75, wherein the
antibody comprises a VH CDR1 having the sequence as set forth in
SEQ ID NO: 13, a VH CDR2 having the sequence as set forth in SEQ ID
NO: 14, a VH CDR3 having the sequence as set forth in SEQ ID NO:
15, a VL CDR1 having the sequence as set forth in SEQ ID NO: 38, a
VL CDR2 having the sequence as set forth in SEQ ID NO: 39, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 40.
87. The combination of nucleic acids of claim 75, wherein the
antibody comprises a VH CDR1 having the sequence as set forth in
SEQ ID NO: 18, a VH CDR2 having the sequence as set forth in SEQ ID
NO: 19, a VH CDR3 having the sequence as set forth in SEQ ID NO:
20, a VL CDR1 having the sequence as set forth in SEQ ID NO: 43, a
VL CDR2 having the sequence as set forth in SEQ ID NO: 44, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 45.
88. The combination of nucleic acids of claim 75, wherein the
antibody comprises a VH CDR1 having the sequence as set forth in
SEQ ID NO: 23, a VH CDR2 having the sequence as set forth in SEQ ID
NO: 24, a VH CDR3 having the sequence as set forth in SEQ ID NO:
25, a VL CDR1 having the sequence as set forth in SEQ ID NO: 48, a
VL CDR2 having the sequence as set forth in SEQ ID NO: 49, and a VL
CDR3 having the sequence as set forth in SEQ ID NO: 50.
89. A combination of expression vectors comprising the combination
of nucleic acids of claim 75.
90. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of expression vectors of claim 89.
91. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 90, and b) purifying
the anti-CD38 antibody from the culture media.
92. An expression vector comprising the combination of nucleic
acids of claim 75.
93. A recombinant eukaryotic or prokaryotic host cell comprising
the expression vector of claim 92.
94. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 93, and b) purifying
the anti-CD38 antibody from the culture media.
95. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of nucleic acids of claim 75.
96. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 95, and b) purifying
the anti-CD38 antibody from the culture media.
97. A combination of nucleic acid encoding an antibody that binds
to CD38, wherein the antibody comprises a VH region comprising any
one of the sequences as set forth in SEQ ID NOs: 2, 7, 12, 17, and
22, and a VL region comprising any one of the sequences as set
forth in SEQ ID NOs: 27, 32, 37, 42, and 47.
98. The combination of nucleic acids of claim 97, wherein the
antibody comprises a VH region comprising the sequence as set forth
in SEQ ID NO: 2, and a VL region comprising the sequence as set
forth in SEQ ID NO: 27.
99. A combination of expression vectors comprising the combination
of nucleic acids of claim 98.
100. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of expression vectors of claim 99.
101. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 100, and b) purifying
the anti-CD38 antibody from the culture media.
102. An expression vector comprising the combination of nucleic
acids of claim 98.
103. A recombinant eukaryotic or prokaryotic host cell comprising
the expression vector of claim 102.
104. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 103, and b) purifying
the anti-CD38 antibody from the culture media.
105. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of nucleic acids of claim 98.
106. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 105, and b) purifying
the anti-CD38 antibody from the culture media.
107. The combination of nucleic acids of claim 97, wherein the
antibody comprises a VH region comprising the sequence as set forth
in SEQ ID NO: 7, and a VL region comprising the sequence as set
forth in SEQ ID NO: 32.
108. The combination of nucleic acids of claim 97, wherein the
antibody comprises a VH region comprising the sequence as set forth
in SEQ ID NO: 12, and a VL region comprising the sequence as set
forth in SEQ ID NO: 37.
109. The combination of nucleic acids of claim 97, wherein the
antibody comprises a VH region comprising the sequence as set forth
in SEQ ID NO: 17, and a VL region comprising the sequence as set
forth in SEQ ID NO: 42.
110. The combination of nucleic acids of claim 97, wherein the
antibody comprises a VH region comprising the sequence as set forth
in SEQ ID NO: 22, and a VL region comprising the sequence as set
forth in SEQ ID NO: 47.
111. A combination of nucleic acids encoding an antibody that binds
to CD38, wherein the antibody comprises: (i) a VH region comprising
the sequence as set forth in SEQ ID NO: 2, and a VL region
comprising the sequence as set forth in SEQ ID NO: 27, (ii) a VH
region comprising the sequence as set forth in SEQ ID NO: 7, and a
VL region comprising the sequence as set forth in SEQ ID NO: 32,
(iii) a VH region comprising the sequence as set forth in SEQ ID
NO: 12, and a VL region comprising the sequence as set forth in SEQ
ID NO: 37, (iv) a VH region comprising the sequence as set forth in
SEQ ID NO: 17, and a VL region comprising the sequence as set forth
in SEQ ID NO: 42, or (v) a VH region comprising the sequence as set
forth in SEQ ID NO: 22, and a VL region comprising the sequence as
set forth in SEQ ID NO: 47.
112. A combination of expression vectors comprising the combination
of nucleic acids of claim 111.
113. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of expression vectors of claim 112.
114. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 113, and b) purifying
the anti-CD38 antibody from the culture media.
115. An expression vector comprising the combination of nucleic
acids of claim 111.
116. A recombinant eukaryotic or prokaryotic host cell comprising
the expression vector of claim 115.
117. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 116, and b) purifying
the anti-CD38 antibody from the culture media.
118. A recombinant eukaryotic or prokaryotic host cell comprising
the combination of nucleic acids of claim 111.
119. A method for producing an anti-CD38 antibody that binds to
human CD38 as set forth in SEQ ID NO: 52, the method comprising the
steps of: a) culturing the host cell of claim 118, and b) purifying
the anti-CD38 antibody from the culture media.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted electronically in ASCII format and is hereby incorporated
by reference in its entirety. Said ASCII copy, created on Mar. 1,
2018, is named GMI_133USDV2_Sequence_Listing.txt and is 26,653
bytes in size.
FIELD OF THE INVENTION
The present invention relates to antibodies directed to human CD38
and to uses of such antibodies, in particular therapeutic uses.
BACKGROUND OF THE INVENTION
CD38 is a type II transmembrane glycoprotein which is normally
found on hemopoietic cells and in solid tissues. With regard to
hemopoietic cells, the majority of medullary thymocytes are
CD38.sup.+, resting and circulating T- and B-cells are CD38.sup.-
and activated cells are CD38.sup.+. CD38 is also expressed on
approximately 80% of resting NK cells and monocytes and on lymph
node germinal center lymphoblasts, plasma B cells and some
intrafollicular cells. CD38 can also be expressed by dendritic
cells. A significant proportion of normal bone marrow cells,
particular precursor cells, express CD38. In addition, 50-80% of
umbilical cord blood cells is CD38.sup.+ and remains so in human
blood for the first two to three years of life. In addition to
lymphoid precursor cells, CD38 is also expressed on erythrocytes
and on platelets. With regard to solid tissues, CD38 is expressed
in the gut by intra-epithelial cells and lamina propria
lymphocytes, by Purkinje cells and neurofibrillary tangles in the
brain, by epithelial cells in the prostate, .beta.-cells in the
pancreas, osteoclasts in the bone, retinal cells in the eye, and
sarcolemma of smooth and striated muscle.
CD38 is also expressed in a variety of malignant hematological
diseases, including multiple myeloma, B-cell chronic lymphocytic
leukemia, B-cell acute lymphocytic leukemia, Waldenstrom
macroglobulinemia, primary systemic amyloidosis, mantle-cell
lymphoma, pro-lymphocytic/myelocytic leukemia, acute myeloid
leukemia, chronic myeloid leukemia, follicular lymphoma, NK-cell
leukemia and plasma-cell leukemia. Expression of CD38 has been
described on epithelial/endothelial cells of different origin,
including glandular epithelium in prostate, islet cells in
pancreas, ductal epithelium in glands, including parotid gland,
bronchial epithelial cells, cells in testis and ovary and tumor
epithelium in colorectal adenocarcinoma. Other diseases, where CD38
expression could be involved, include, e.g. broncho-epithelial
carcinomas of the lung, breast cancer (evolving from malignant
proliferation of epithelial lining in ducts and lobules of the
breast), pancreatic tumors, evolving from the b-cells
(insulinomas), tumors evolving from epithelium in the gut (e.g.
adenocarcinoma and squamous cell carcinoma), carcinoma in the
prostate gland, seminomas in testis and ovarian cancers. In CNS,
neuroblastomas express CD38.
Other disclosures also suggest the role of CD38 in autoimmunity
such as Graves disease and thyroiditis (Antonelli A, et. al., Clin.
Exp. Immunol. 126, 426-431, 2001), and type 1 and 2 Diabetes
(Mallone R and Perin PC, Diabetes Metab Res Rev 2006; 22: 284-294)
and inflammation of airway smooth muscle cells during asthma
(Desphande et al. 2004 am J Respir Cell Mol Biol 31: 36-42)
CD38 is a multifunctional protein. Functions ascribed to CD38
include both receptor mediation in adhesion and signaling events
and (ecto-) enzymatic activity. As an ectoenzyme, CD38 uses NAD as
substrate for the formation of cyclic ADP-ribose (cADPR) and ADPR,
but also of nicotinamide and nicotinic acid-adenine dinucleotide
phosphate (NAADP). cADPR has been shown to act as second messenger
for Ca.sup.2+ mobilization from the endoplasmatic reticulum. The
CD38/cyclic ADP ribose system: 1) in lung, contributes to airway
smooth muscle tone and responsiveness through its effects on
agonist induced elevation of intra-cellular Ca.sup.2+ (Desphande et
al. 2005 Am J physiol Lung cell Mol Physiol 288: L773-L788), 2)
regulates migration of neutrophil chemotaxis to bacterial
chemoattractants, migration of DC precursors from blood to
peripheral sites and migration of mature DCs from sites of
inflammation to lymph nodes (Partida-Sanchez et al. Nat Med 7:
1209-121, 2001; Morita et al. 2008 J Pharmacol Sci. 2008
Mar;106(3):492-504; Partida-Sanchez et al. Immunity 20: 279-291,
2004), 3) is involved in astrocyte calcium signaling which has
implications for neuroinflammation and HIV-1-associated dementia
(Banerjee S. et. al., J. Neurimmune Pharmacol., 3, 154-164 (2008)),
4) regulates Fc.gamma.R-mediated phagocytosis in murine macrophages
(Song E., et. al., Biochem. and Biophys. Res. Comm., 367, 156-161,
(2008), 5) is linked to insulin secretion Okamoto, Molecular and
Cellular Biochemistry, 193, 115-118, 1999 and 6) has a key role in
neuropeptide release and regulating maternal and social behaviors
(Jin D et al. Nature 446: 41-45, 2007). In addition to signaling
via Ca.sup.2+, CD38 signaling occurs via cross-talk with
antigen-receptor complexes on T and B cells or other types of
receptor complexes, e.g. MHC molecules, and is in this way involved
in several cellular responses, but also in switching and secretion
of IgG1.
Several anti-CD38 antibodies are described in the literature, for
instance in Lande R, et al., Cell Immunol. 220(1), 30-8 (2002),
Ausiello CM, et al., Tissue Antigens. 56(6), 539-47 (2000), and
Cotner T, et al., Int J Immunopharmacol. 3(3), 255-68 (1981).
Antibody binding to CD38 can have different effects on the
functions of CD38. For instance, mouse anti-CD38 antibody IB4 has
been shown to induce T cell activation as indicated by Ca.sup.2+
mobilization in Jurkat cells (Zubiaur M, et al., J Immunol. 159(1),
193-205 (1997), to induce significant proliferation of peripheral
blood mononuclear cells (PBMCs), to induce release of significant
IL-6 levels and to induce release of detectable IFN-.gamma. levels
(Lande, Zubiaur Morra, Ansiello supra). Hara-Yokoyama et al. Int
Immunopharmacol 8, 59-70 (2008) described one anti-mouse CD38
antibody (CS/2) which inhibits the NAD glycohydrolase activity of
CD38 and another anti-mouse CD38 antibody (clone 90) which
stimulates the NAD.sup.+ glycohydrolase activity of an isolated
extracellular domain of CD38, but has little effect on the
NAD.sup.+ glycohydrolase activity of cell-surface CD38. As it can
be seen from data presented below, the antibodies of the present
invention provide activity on the surface of CD38 positive
cells.
WO2006099875 (Genmab) describes several human anti-CD38 antibodies,
including 003 and 005. Antibody 005 was shown to inhibit the
production of cGDPR from NGD.sup.+ by CD38.
In view of the multiple functions of human CD38, there is a need
for new therapeutic antibodies that more specifically modulate
particular functions of CD38.
SUMMARY OF THE INVENTION
The present invention provides a new class of anti-CD38 antibodies
which through interacting with particular amino acids of human CD38
have a strong stimulating effect on the cADPR hydrolase activity of
CD38 leading to decreased levels of cADPR. Furthermore, the
anti-CD38 antibodies inhibit the ability of CD38 to catalyze the
formation, via a base-exchange reaction, of nicotinic acid adenine
dinucleotide 2'-phosphate (NAADP).
These antibodies are useful for the treatment of several diseases,
including autoimmune and (chronic) inflammatory diseases, such as
Type 1 and 2 diabetes, thyroiditis, Graves disease, arthritis,
neuroinflammation and asthma.
Recent scientific work suggests that cADPR synthesized
extracellularly by CD38, may be transported into cells through
nucleoside transporters and then mobilize Ca(2+) through a
FK506-binding protein-dependent process. This process may be
involved in fMLP-induced intracellular Ca(2+) signaling and
migration in human neutrophils (Morita et al. 2008 J Pharmacol Sci.
2008 Mar;106(3):492-504), migration of DC precursors from blood to
peripheral sites and migration of mature DCs from sites of
inflammation to lymph nodes (Partida-Sanchez et al. Immunity 20:
279-291, 2004). Without being bound by any particular theory, the
reduction of cADPR levels obtained by treatment with an antibody of
the present invention may thus reduce migration of neutrophils and
dendritic cells and have anti-inflammatory effects. Accordingly,
while the antibodies of the invention may be useful for a number of
purposes, they may be particularly useful for the treatment of
inflammation, e.g. in connection with autoimmune disease, because
of their unique effects on the enzymatic activities of CD38,
through binding at a particular site on CD38.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows cross-block studies of antibodies of the invention.
More particularly, the figure shows the binding of 005-FITC to
CHO-CD38 cells treated with excess unlabelled CD38-specific
antibodies 025, 026, 028, 049 and 056.
FIGS. 2A and 2B show binding of the anti-CD38 antibodies of the
invention to wt and mutant CD38. FIG. 2A shows binding of yhe
anti-CD38 antibodies 025, 026, 028 and 049 to wild type (WT) and
mutant (T237A, Q272R, and S274F) CD38. FIG. 2B shows binding of the
anti-CD38 antibodies 025, 028 and 049 to wild type (WT) and mutant
(D202G) CD38.
FIG. 3 shows binding of antibodies of the invention to Daudi-luc
cells and CHO-CD38 cells.
FIG. 4 shows ADCC mediated lysis of Daudi-luc cells caused by the
anti-CD38 antibodies of the invention and as isotype control
anti-KLH antibody (HuMab-KLH).
FIG. 5 shows CDC mediated lysis of CHO CD38 cells caused by the
anti-CD38 antibodies of the invention.
FIGS. 6A-6D show inhibition of cGDPR production by His-tagged CD38
protein and cellular expressed CD38 in the presence of the
anti-CD38 antibodies of the invention. FIG. 6A shows the percentage
inhibition of cGDPR production (by recombinant human CD38 protein)
in the presence of CD38 specific antibodies 025, 026, 028, 049 and
056 (3 .mu.g/mL). FIG. 6B shows the effect of the anti-CD38
antibodies on cGDPR production in time. The anti-CD38 antibodies
were used at a final concentration of 10 .mu.g/ml. FIG. 6C shows
the effect of the anti-CD38 antibodies on cGDPR production using
serial dilutions (0.01-30 .mu.g/mL) of 028 or isotype
controlHuMab-KLH. FIG. 6D shows the percentage inhibition of cGDPR
production (by cellular expressed CD38 (CHO-CD38 cells)) in the
presence of serial dilutions (0.01-30 .mu.g/mL) of 028 or IgG1
isotype control HuMab-KLH.
FIGS. 7A and 7B show the effect of antibody 028 of the invention on
8NH2-cADPR production. Products of each reaction were analyzed by
HPLC. FIG. 7A indicates the elution position of the products and
substrates. FIG. 7B shows the antibody concentration dependence on
8NH.sub.2-cADPR production. HuMab-KLH (open circles), mAb-028
(closed circles).
FIGS. 8A-8C show the effect of antibody 028 of the invention on
cADPR hydrolase and NADa se activity, more particularly the effect
of mAb-028 on cADPR hydrolase (FIG. 8A, left figure, FIGS. 8B and
8C) and NADse (FIG. 8A, right figure) activity. FIG. 8A shows the
results of incubating CD38 recombinant protein with cADPR or NAD in
the presence of 10 .mu.g HuMab-KLH (CD38+10 .mu.g HuMab-KLH), 10
.mu.g Ab 028 (CD38+10 .mu.g Ab 028), or no antibody (CD38 control).
Products of each reaction were analyzed by HPLC. FIG. 8B shows CD38
antibodidy titration at different concentrations on cADPR hydrolase
activity as analyzed by HPLC. FIG. 8C shows the results of
incubating CD38 recombinant protein with .sup.32P-cADPR in the
presence of mAb-003, mAb-028, daratumumab (005), or HuMab-KLH.
Products were analyzed by thin layer chromatography. HuMab-KLH
(open circles), mAb-028 (closed circles).
FIGS. 9A and 9B show the effect of the anti-CD38 antibodies of the
invention on the base-exchange activity of CD38. FIG. 9A shows the
effect of the antibodies on NAADP production at the indicated
concentrations. FIG. 9B shows the effect of mAb-028 titration on
the rate of NAADP formation.
TABLE-US-00001 SEQUENCE LIST VH-region SEQ ID NO: 1 VH 028 DNA
caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
tcctgcaagg cttttggagg caccttcagc agctacgcta tcagctgggt gcgacaggcc
cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtat agcaaactac
gcacagaagt tccagggcag agtcacgctt atcgcggaca aatccacgaa cacagcctac
atggagctga gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct
accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct tca SEQ ID
NO: 2 VH 028 QVQLVQSGAE VKKPGSSVKV SCKAFGGTFS SYAISWVRQA PGQGLEWM
GR IIRFLGIANYAQKFQGRVTL IADKSTNTAY MELSSLRSED TAVYYCAGEP GERDPDAVDI
WGQGTMVTVSS SEQ ID NO: 3 VH 028 CDR1 GGTSFSSYA SEQ ID NO: 4 VH 028
CDR2 IIRFLGIA SEQ ID NO: 5 VH 028 CDR3 AGEPGERDPDAVDI SEQ ID NO: 6
VH 025 DNA
caggtccaactggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtc
tcctgcaaggcttttggaggcaccttcagcagctatgctatcagctgggtacgacaggcc
cctggacaagggcttgagtggatgggaaggatcatccgtttccttggtaaagcaaatcac
gcacagaagttccagggcagagtcacgcttaccgcggacaaatccacgaacacagcctac
atggagctgagcagcctgagatctgaggacacggccgtttattactgtgcgggggaacct
ggggatcgggaccccgatgctgttgatatctggggccaagggacaatggtcaccgtctct tcag
SEQ ID NO: 7 VH 025
QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGKANH
AQKFQGRVTLTADKSTNTAYMELSSLRSEDTAVYYCAGEPGDRDPDAVDIWGQGTMVTVS S SEQ
ID NO: 8 VH 025 CDR1 GGTFSSYA SEQ ID NO: 9 VH 025 CDR2 IIRFLGKA SEQ
ID NO: 10 VH 025 CDR3 AGEPGDRDPDAVDI SEQ ID NO: 11 VH 026 DNA
caggtccaactggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtc
tcctgcaaggcttttggaggcaccttcagcagttatgctattagctgggtgcgacaggcc
cctggacaagggcttgagtggatgggaaggatcatccgtttccttggtaaaacaaatcac
gcacagaagttccagggcagagtcacacttaccgcggacaaatccacgaacacagcctac
atggagctgagcagcctgagatctgaggacacggccgtttattactgtgcgggggaacct
ggggatcgggaccccgatgctgttgatatctggggccaagggacaatggtcaccgtctct tcag
SEQ ID NO: 12 VH 026
QVQLVQSGAEVKKPGSSVKVSCKAFGGTFSSYAISWVRQAPGQGLEWMGRIIRFLGKTNH
AQKFQGRVTLTADKSTNTAYMELSSLRSEDTAVYYCAGEPGDRDPDAVDIWGQGTMVTVS S SEQ
ID NO: 13 VH 026 CDR1 GGTFSSYA SEQ ID NO: 14 VH 026 CDR2 IIRFLGKT
SEQ ID NO: 15 VH 026 CDR3 AGEPGDRDPDAVDI SEQ ID NO: 16 VH 049 DNA
caggtccagctggtgcagtctggggctgaggtgatgaagcctgggtcctcggtgaaggtc
tcctgcaaggcttccggaggcaccttccgcagctatgctatcagttgggtgcgacaggcc
cctggacaagggcttgagtggatgggaaggatcatcgttttccttggtaaaacaaactac
gcacagaagttccagggcagagtcacgcttaccgcggacaaatccacgaccacagcctac
atggagctgagcagcctgagatctgaggacacggccgtgtattactgtacgggggaacct
ggggctcgggaccccgacgcttttgatatctggggccaagggacaatggtcaccgtctct tcag
SEQ ID NO: 17 VH 049
QVQLVQSGAEVMKPGSSVKVSCKASGGTFRSYAISWVRQAPGQGLEWMGRIIVFLGKTNY
AQKFQGRVTLTADKSTTTAYMELSSLRSEDTAVYYCTGEPGARDPDAFDIWGQGTMVTVS S SEQ
ID NO: 18 VH 049 CDR1 GGTFRSYA SEQ ID NO: 19 VH 049 CDR2 IIVFLGKT
SEQ ID NO: 20 VH 049 CDR3 TGEPGARDPDAFDI SEQ ID NO: 21 VH 056 DNA
caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtc
tcctgcaagccttccggaggcaccttcaggagctacgctatcagctgggtacgacaggcc
cctggacaagggcttgagtggatgggaaggatcatcgttttccttggtaaagtaaactac
gcacagaggtttcagggcagagtcacgcttaccgcggacaaatccacgaccacagcctac
atggagctgagcagcctgagatctgaggacacggccgtgtattactgtacgggggaacct
ggggctcgggaccccgacgcttttgatatctggggccaagggacaatggtcaccgtctct tcag
SEQ ID NO: 22 VH 056
QVQLVQSGAEVKKPGSSVKVSCKPSGGTFRSYAISWVRQAPGQGLEWMGRIIVFLGKVNY
AQRFQGRVTLTADKSTTTAYMELSSLRSEDTAVYYCTGEPGARDPDAFDIWGQGTMVTVS S SEQ
ID NO: 23 VH 056 CDR1 GGTFRSYA SEQ ID NO: 24 VH 056 CDR2 IIVFLGKV
SEQ ID NO: 25 VH 056 CDR3 TGEPGARDPDAFDI SEQ ID NO: 26 VL 028 DNA
gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc
atcacttgtcgggcgagtcagggtattcgcagctggttagcctggtatcagcagaaacca
gagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatca
aggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct
gaagattttgcaacttattactgccaacagtataatagttacccgctcactttcggcgga
gggaccaaggtggagatcaaa SEQ ID NO: 27 VL 028
DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID NO: 28 VL
028 CDR1 GGIRSW SEQ ID NO: 29 VL 028 CDR2 AAS SEQ ID NO: 30 VL 028
CDR3 QQYNSYPLT SEQ ID NO: 31 VL 025 DNA
gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc
atcacttgtcgggcgagtcagggtattcgcagctggttagcctggtatcagcagaaacca
gagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatca
aggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct
gaagattttgcaacttattactgccaacagtataatagttacccgctcactttcggcgga
gggaccaaggtggagatcaaac SEQ ID NO: 32 VL 025
DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID NO: 33 VL
025 CDR1 QGIRSW SEQ ID NO: 34 VL 025 CDR2 AAS SEQ ID NO: 35 VL 025
CDR3 QQYNSYPLT SEQ ID NO: 36 VL 026 DNA
gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc
atcacttgtcgggcgagtcagggtattcgcagctggttagcctggtatcagcagaaacca
gagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatca
aggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct
gaagattttgcaacttattactgccaacagtataatagttacccgctcactttcggcgga
gggaccaaggtggagatcaaac SEQ ID NO: 37 VL 026
DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGGGTKVEIK SEQ ID NO: 38 VL
026 CDR1 QGIRSW SEQ ID NO: 39 VL 026 CDR2 AAS SEQ ID NO: 40 VL 026
CDR3 QQYNSYPLT SEQ ID NO: 41 VL 049 DNA
gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc
atcacttgtcgggcgagtcagggtattcgcagctggttagcctggtatcagcagaaacca
gagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatca
aggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct
gaagattttgcaacttattactgccaacagtataataattatccgctcactttcggcgga
gggaccaaggtggagatcaaac SEQ ID NO: 42 VL 049
DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK SEQ ID NO: 43 VL
049 CDR1 QGIRSW SEQ ID NO: 44 VL 049 CDR2 AAS SEQ ID NO: 45 VL 049
CDR3 QQYNNYPLT SEQ ID NO: 46 VL 056 DNA
gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcacc
atcacttgtcgggcgagtcagggtattcgcagctggttagcctggtatcagcagaaacca
gagaaagcccctaagtccctgatctatgctgcatccagtttgcaaagtggggtcccatca
aggttcagcggcagtggatctgggacagatttcactctcaccatcagcagcctgcagcct
gaagattttgcaacttattactgccaacagtataataattatccgctcactttcggcgga
gggaccaaggtggagatcaaac SEQ ID NO: 47 VL 056
DIQMTQSPSSLSASVGDRVTITCRASQGIRSWLAWYQQKPEKAPKSLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPLTFGGGTKVEIK SEQ ID NO: 48 VL
056 CDR1 QGIRSW SEQ ID NO: 49 VL 056 CDR2 AAS SEQ ID NO: 50 VL 056
CDR3 QQYNNYPLT SEQ ID NO: 51 Mutant human CD38
MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRF
PETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVP
CNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWR
KDCSNNPVSVFWKTVSRRFAEAACGVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQ
TLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSS CTSEI
SEQ ID NO: 52 Human CD38
MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRF
PETVLARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVP
CNKILLWSRIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWR
KDCSNNPVSVFWKTVSRRFAEAACDVVHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQ
TLEAWVIHGGREDSRDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSS
CTSEI
CDR regions are indicated according to IMGT.
The sequence of human CD38 is described in sequence 52. A mutant of
human CD38 wherein S was mutated to F at position 274 was described
in W02006099875 as SEQ. ID NO: 34, and a mutation wherein Q was
mutated to R at position 272 was described in WO2006099875 as SEQ.
ID NO: 33. A mutant of human CD38 wherein D was mutated to G iat
position 202 is described above as SEQ. ID NO: 51.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "human CD38" when used herein includes any variants,
isoforms and species homologs of human CD38 (Swissprot: locus
CD38_HUMAN, accession P28907) which are naturally expressed by
cells or are expressed on cells transfected with the human CD38
gene.
The term "immunoglobulin" refers to a class of structurally related
glycoproteins consisting of two pairs of polypeptide chains, one
pair of light (L) low molecular weight chains and one pair of heavy
(H) chains, all four inter-connected by disulfide bonds. The
structure of immunoglobulins has been well characterized. See for
instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven
Press, N.Y. (1989)). Briefly, each heavy chain typically is
comprised of a heavy chain variable region (abbreviated herein as
VH or VH) and a heavy chain constant region. The heavy chain
constant region typically is comprised of three domains, C.sub.H1,
C.sub.H2, and C.sub.H3. Each light chain typically is comprised of
a light chain variable region (abbreviated herein as V.sub.L or VL)
and a light chain constant region. The light chain constant region
typically is comprised of one domain, C.sub.L. The V.sub.H and
V.sub.L regions may be further subdivided into regions of
hypervariability (or hypervariable regions which may be
hypervariable in sequence and/or form of structurally defined
loops), also termed complementarity determining regions (CDRs),
interspersed with regions that are more conserved, termed framework
regions (FRs). Each V.sub.H and V.sub.L is typically composed of
three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917
(1987)). Typically, the numbering of amino acid residues in this
region is performed by the method described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md. (1991)
(phrases such as variable domain residue numbering as in Kabat or
according to Kabat herein refer to this numbering system for heavy
chain variable domains or light chain variable domains). Using this
numbering system, the actual linear amino acid sequence of a
peptide may contain fewer or additional amino acids corresponding
to a shortening of, or insertion into, a FR or CDR of the variable
domain. For example, a heavy chain variable domain may include a
single amino acid insert (residue 52a according to Kabat) after
residue 52 of V.sub.H CDR2 and inserted residues (for instance
residues 82a, 82b, and 82c, etc. according to Kabat) after heavy
chain FR residue 82. The Kabat numbering of residues may be
determined for a given antibody by alignment at regions of homology
of the sequence of the antibody with a "standard" Kabat numbered
sequence.
The term "antibody" (Ab) in the context of the present invention
refers to an immunoglobulin molecule, a fragment of an
immunoglobulin molecule, or a derivative of either thereof, which
has the ability to specifically bind to an antigen under typical
physiological conditions with a half life of significant periods of
time, such as at least about 30 minutes, at least about 45 minutes,
at least about one hour, at least about two hours, at least about
four hours, at least about 8 hours, at least about 12 hours, about
24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or
more days, etc., or any other relevant functionally-defined period
(such as a time sufficient to induce, promote, enhance, and/or
modulate a physiological response associated with antibody binding
to the antigen and/or time sufficient for the antibody to recruit
an Fc-mediated effector activity). The variable regions of the
heavy and light chains of the immunoglobulin molecule contain a
binding domain that interacts with an antigen. The constant regions
of the antibodies (Abs) may mediate the binding of the
immunoglobulin to host tissues or factors, including various cells
of the immune system (such as effector cells) and components of the
complement system such as C1q, the first component in the classical
pathway of complement activation. An anti-CD38 antibody may also be
a bispecific antibody, diabody, or similar molecule (see for
instance PNAS USA 90(14), 6444-8 (1993) for a description of
diabodies). Indeed, bispecific antibodies, diabodies, and the like,
provided by the present invention may bind any suitable target in
addition to a portion of CD38. As indicated above, the term
antibody herein, unless otherwise stated or clearly contradicted by
context, includes fragments of an antibody that retain the ability
to specifically bind to the antigen. It has been shown that the
antigen-binding function of an antibody may be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antibody" include (i) a Fab' or Fab
fragment, a monovalent fragment consisting of the V.sub.L, V.sub.H,
C.sub.L and C.sub.H1 domains, or a monovalent antibody as described
in WO2007059782 (Genmab); (ii) F(ab').sub.2 fragments, bivalent
fragments comprising two Fab fragments linked by a disulfide bridge
at the hinge region; (iii) a Fd fragment consisting essentially of
the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment consisting
essentially of the V.sub.L and V.sub.H domains of a single arm of
an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546
(1989)), which consists essentially of a V.sub.H domain and also
called domain antibodies (Holt et al; Trends Biotechnol. 2003
Nov;21(11):484-90); (vi) camelid or nanobodies (Revets et al;
Expert Opin Biol Ther. 2005 Jan;5(1):111-24) and (vii) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, V.sub.L and V.sub.H, are coded for
by separate genes, they may be joined, using recombinant methods,
by a synthetic linker that enables them to be made as a single
protein chain in which the V.sub.L and V.sub.H regions pair to form
monovalent molecules (known as single chain antibodies or single
chain Fv (scFv), see for instance Bird et al., Science 242, 423-426
(1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such
single chain antibodies are encompassed within the term antibody
unless otherwise noted or clearly indicated by context. Although
such fragments are generally included within the meaning of
antibody, they collectively and each independently are unique
features of the present invention, exhibiting different biological
properties and utility. These and other useful antibody fragments
in the context of the present invention are discussed further
herein. It also should be understood that the term antibody, unless
specified otherwise, also includes polyclonal antibodies,
monoclonal antibodies (mAbs), antibody-like polypeptides, such as
chimeric antibodies and humanized antibodies, and antibody
fragments retaining the ability to specifically bind to the antigen
(antigen-binding fragments) provided by any known technique, such
as enzymatic cleavage, peptide synthesis, and recombinant
techniques. An antibody as generated can possess any isotype. As
used herein, "isotype" refers to the immunoglobulin class (for
instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that is
encoded by heavy chain constant region genes. An "anti-CD38
antibody" is an antibody which binds to the antigen CD38.
The term "human antibody", as used herein, is intended to include
antibodies having variable and constant regions derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). However, the term "human antibody", as used herein, is
not intended to include antibodies in which CDR sequences derived
from the germline of another mammalian species, such as a mouse,
have been grafted onto human framework sequences.
In a preferred embodiment, the antibody of the invention is
isolated. An "isolated antibody," as used herein, is intended to
refer to an antibody which is substantially free of other
antibodies having different antigenic specificities (for instance
an isolated antibody that specifically binds to CD38 is
substantially free of antibodies that specifically bind antigens
other than CD38). An isolated antibody that specifically binds to
an epitope, isoform or variant of human CD38 may, however, have
cross-reactivity to other related antigens, for instance from other
species (such as CD38 species homologs). Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals. In one embodiment of the present invention, a
combination of "isolated" monoclonal antibodies having different
specificities is combined in a well-defined composition.
The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions derived from
human germline immunoglobulin sequences. The human monoclonal
antibodies may be generated by a hybridoma which includes a B cell
obtained from a transgenic or transchromosomal nonhuman animal,
such as a transgenic mouse, having a genome comprising a human
heavy chain transgene and a light chain transgene, fused to an
immortalized cell.
As used herein, the term "binding" in the context of the binding of
an antibody to a predetermined antigen typically is a binding with
an affinity corresponding to a K.sub.D of about 10.sup.-7 M or
less, such as about 10.sup.-8 M or less, such as about 10.sup.-9 M
or less, about 10.sup.-10 M or less, or about 10.sup.-11 M or even
less when determined by for instance surface plasmon resonance
(SPR) technology in a BIAcore 3000 instrument using the antigen as
the ligand and the antibody as the analyte, and binds to the
predetermined antigen with an affinity corresponding to a K.sub.D
that is at least ten-fold lower, such as at least 100 fold lower,
for instance at least 1,000 fold lower, such as at least 10,000
fold lower, for instance at least 100,000 fold lower than its
affinity for binding to a non-specific antigen (e.g., BSA, casein)
other than the predetermined antigen or a closely-related antigen.
The amount with which the affinity is lower is dependent on the
K.sub.D of the antibody, so that when the K.sub.D of the antibody
is very low (that is, the antibody is highly specific), then the
amount with which the affinity for the antigen is lower than the
affinity for a non-specific antigen may be at least 10,000
fold.
The term "k.sub.d" (sec.sup.-1), as used herein, refers to the
dissociation rate constant of a particular antibody-antigen
interaction. Said value is also referred to as the k.sub.off
value.
The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used herein,
refers to the association rate constant of a particular
antibody-antigen interaction.
The term "K.sub.D" (M), as used herein, refers to the dissociation
equilibrium constant of a particular antibody-antigen
interaction.
The term "K.sub.A" (M.sup.-1), as used herein, refers to the
association equilibrium constant of a particular antibody-antigen
interaction and is obtained by dividing the k.sub.a by the
k.sub.d.
The antibodies of the present invention have an effect on enzymatic
systems as described in the examples section. The antibodies are
described by stimulatory effects or inhibitory effects on different
parameters. The stimulatory and inhibitory effects may be measured
as disclosed in the examples herein.
An antibody as described and claimed herein may also be a
functional variant of any of the specific antibodies described
herein. Such a variant antibody is an antibody that differs from a
specific antibody described herein by one or more suitable amino
acid residue alterations, that is substitutions, deletions,
insertions, or terminal sequence additions, for instance in the
constant domain, and/or the variable regions (or any one or more
CDRs thereof) in a single variant antibody. A functional variant of
a V.sub.L, V.sub.H, or CDR region used in the context of an
anti-CD38 antibody still allows the antibody to retain at least a
substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%
or more) of the affinity/avidity and/or the specificity/selectivity
of the parent antibody and in some cases such an anti-CD38 antibody
may be associated with greater affinity, selectivity and/or
specificity than the parent antibody.
Such functional variants typically retain significant sequence
identity to the parent antibody. The percent identity between two
sequences is a function of the number of identical positions shared
by the sequences (i.e., % homology=# of identical positions/total #
of positions.times.100), taking into account the number of gaps,
and the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences may be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
The percent identity between two nucleotide sequences may be
determined using the GAP program in the GCG software package
(available at http://www.gcg.com), using a NWSgapdna.CMP matrix and
a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5, or 6. The percent identity between two nucleotide or amino
acid sequences may also be determined using the algorithm of E.
Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988)) which
has been incorporated into the ALIGN program (version 2.0), using a
PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty of 4. In addition, the percent identity between two amino
acid sequences may be determined using the Needleman and Wunsch, J.
Mol. Biol. 48, 444-453 (1970)) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at http://www.gcg.com), using either a Blossum 62 matrix
or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4
and a length weight of 1, 2, 3, 4, 5, or 6.
The sequence of CDR variants may differ from the sequence of the
CDR of the parent antibody sequences through mostly conservative
substitutions; for instance at least about 35%, about 50% or more,
about 60% or more, about 70% or more, about 75% or more, about 80%
or more, about 85% or more, about 90% or more, about 95% or more
(e.g., about 65-99%) of the substitutions in the variant are
conservative amino acid residue replacements. In the context of the
present invention, conservative substitutions may be defined by
substitutions within the classes of amino acids reflected in one or
more of the following three tables:
Amino Acid Residue Classes for Conservative Substitutions
TABLE-US-00002 Acidic Residues Asp (D) and Glu (E) Basic Residues
Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser
(S), Thr (T), Asn (N), and Gln (Q) Aliphatic Uncharged Residues Gly
(G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar Uncharged
Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F),
Tyr (Y), and Trp (W)
Alternative Conservative Amino Acid Residue Substitution
Classes
TABLE-US-00003 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W
Alternative Physical and Functional Classifications of Amino Acid
Residues
TABLE-US-00004 Alcohol group-containing S and T residues Aliphatic
residues I, L, V, and M Cycloalkenyl-associated F, H, W, and Y
residues Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W,
and Y Negatively charged D and E residues Polar residues C, D, E,
H, K, N, Q, R, S, and T Positively charged H, K, and R residues
Small residues A, C, D, G, N, P, S, T, and V Very small residues A,
G, and S Residues involved in A, C, D, E, G, H, K, turn formation
N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E, and
R
More conservative substitutions groupings include:
valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,
alanine-valine, and asparagine-glutamine.
Additional groups of amino acids may also be formulated using the
principles described in, e.g., Creighton (1984) Proteins: Structure
and Molecular Properties (2d Ed. 1993), W.H. Freeman and
Company.
As explained above, typically, amino acid sequence alterations,
desirably do not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to disrupt secondary structure that
characterizes the function of the parent sequence), but may be
associated with advantageous properties, such as changing the
functional or pharmacokinetic properties of the antibodies, for
example increasing the half-life, altering the immunogenicity,
providing a site for covalent or non-covalent binding to another
molecule, reducing susceptibility to proteolysis, reducing
susceptibility to oxidation, or altering the glycosylation
pattern.
Examples of functional properties of antibodies, which may be
altered or retained in variant anti-CD38 antibodies of the present
invention compared to antibodies of prior art are for example: (1)
high affinity binding to CD38 and/or (2) binding to transfected
cells, e.g. CHO or HEK293 cells expressing CD38 and/or (3)
induction of CDC and/or (4) induction of ADCC and/or (5) alteration
of enzymatic activity and/or (6) induction of apoptosis after
secondary cross-linking and/or (7) phagocytosis
The term "epitope" means a protein determinant capable of specific
binding to an antibody. Epitopes usually consist of surface
groupings of molecules such as amino acids or sugar side chains and
usually have specific three dimensional structural characteristics,
as well as specific charge characteristics. Conformational and
nonconformational epitopes are distinguished in that the binding to
the former but not the latter is lost in the presence of denaturing
solvents. The epitope may comprise amino acid residues directly
involved in the binding (also called immunodominant component of
the epitope) and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide (in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide).
As used herein, a human antibody is "derived from" a particular
germline sequence if the antibody is obtained from a system using
human immunoglobulin sequences, for instance by immunizing a
transgenic mouse carrying human immunoglobulin genes or by
screening a human immunoglobulin gene library, and wherein the
selected human antibody is at least 90%, such as at least 95%, for
instance at least 96%, such as at least 97%, for instance at least
98%, or such as at least 99% identical in amino acid sequence to
the amino acid sequence encoded by the germline immunoglobulin
gene. Typically, outside the heavy chain CDR3, a human antibody
derived from a particular human germline sequence will display no
more than 20 amino acid differences, e.g. no more than 10 amino
acid differences, such as no more than 5, for instance no more than
4, 3, 2, or 1 amino acid difference from the amino acid sequence
encoded by the germline immunoglobulin gene.
As used herein, the term "inhibits growth" (e.g. referring to
cells, such as tumor cells) is intended to include any measurable
decrease in the cell growth when contacted with an anti-CD38
antibody as compared to the growth of the same cells not in contact
with an anti-CD38 antibody, e.g., the inhibition of growth of a
cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 99%, or 100%. Such a decrease in cell growth can occur by
a variety of mechanisms, e.g. effector cell phagocytosis, ADCC,
CDC, and/or apoptosis.
The term "bispecific antibody" is intended to include any antibody
which has two different binding specificities. The term "bispecific
antibodies" also includes diabodies (see for instance Holliger, P.
et al., PNAS USA 90, 6444-6448 (1993), Poljak, R. J. et al.,
Structure 2, 1121-1123 (1994)).
An "antibody deficient in effector function" or an
"effector-function-deficient antibody" refers to an antibody which
has a significantly reduced or no ability to activate one or more
immune effector mechanisms, such as complement activation or Fc
receptor binding. Thus, effector-function deficient antibodies have
significantly reduced or no ability to mediate antibody-dependent
cell-mediated cytotoxicity (ADCC) and/or complement-dependent
cytotoxicity (CDC).
The term "monovalent antibody" means in the context of the present
invention that an antibody molecule is capable of binding a single
molecule of the antigen, and thus is not able of antigen
crosslinking.
As used herein, the term "effector cell" refers to an immune cell
which is involved in the effector phase of an immune response, as
opposed to the cognitive and activation phases of an immune
response. Exemplary immune cells include a cell of a myeloid or
lymphoid origin, for instance lymphocytes (such as B cells and T
cells including cytolytic T cells (CTLs)), killer cells, natural
killer cells, macrophages, monocytes, eosinophils,
polymorphonuclear cells, such as neutrophils, granulocytes, mast
cells, and basophils. Some effector cells express specific Fc
receptors and carry out specific immune functions. In some
embodiments, an effector cell is capable of inducing
antibody-dependent cellular cytotoxicity (ADCC), such as a natural
killer cell, capable of inducing ADCC. For example, monocytes,
macrophages, which express FcR are involved in specific killing of
target cells and presenting antigens to other components of the
immune system, or binding to cells that present antigens. In some
embodiments, an effector cell may phagocytose a target antigen or
target cell.
The term "vector," as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. Various types of vectors are
well-known in the art. One type of vector is a plasmid.
The term "recombinant host cell" (or simply "host cell"), as used
herein, is intended to refer to a cell into which an expression
vector has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell, but
also to the progeny of such a cell. Because certain modifications
may occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. Recombinant host
cells include, for example, transfectomas, such as CHO cells,
HEK293 cells, NS/0 cells, and lymphocytic cells.
The term "transgenic non-human animal" refers to a non-human animal
having a genome comprising one or more human heavy and/or light
chain transgenes or transchromosomes (either integrated or
non-integrated into the animal's natural genomic DNA) and which is
capable of expressing fully human antibodies. For example, a
transgenic mouse can have a human light chain transgene and either
a human heavy chain transgene or human heavy chain transchromosome,
such that the mouse produces human anti-CD38 antibodies when
immunized with CD38 antigen and/or cells expressing CD38. The human
heavy chain transgene may be integrated into the chromosomal DNA of
the mouse, as is the case for transgenic mice, for instance HuMAb
mice, such as HCo7 or HCo12 mice, or the human heavy chain
transgene may be maintained extrachromosomally, as is the case for
transchromosomal KM mice as described in WO02/43478. Such
transgenic and transchromosomal mice (collectively referred to
herein as "transgenic mice") are capable of producing multiple
isotypes of human monoclonal antibodies to a given antigen (such as
IgG, IgA, IgM, IgD and/or IgE) by undergoing V-D-J recombination
and isotype switching. Transgenic, nonhuman animal can also be used
for production of antibodies against a specific antigen by
introducing genes encoding such specific antibody, for example by
operatively linking the genes to a gene which is expressed in the
milk of the animal.
The terms "B-cell neoplasms" or "mature B-cell neoplasms" in the
context of the present invention include small lymphocytic
lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic
lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma,
follicular lymphoma, diffuse large B-cell lymphoma, multiple
myeloma, lymphoplasmacytic lymphoma, splenic margina zone lymphoma,
plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition disease, heavy chain disease,
MALT lymphoma, nodal marginal B cell lymphoma, intravascular large
B cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy
cell leukemia, primary effusion lymphoma and AIDS-related
non-Hodgkins lymphoma.
"Treatment" refers to the administration of an effective amount of
a therapeutically active compound of the present invention with the
purpose of easing, ameliorating, arresting or eradicating (curing)
symptoms or disease states.
An "effective amount" refers to an amount effective, at dosages and
for periods of time necessary, to achieve a desired therapeutic
result. A therapeutically effective amount of an anti-CD38 antibody
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the anti-CD38
antibody to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the antibody or antibody portion are
outweighed by the therapeutically beneficial effects.
An "anti-idiotypic" (Id) antibody is an antibody which recognizes
unique determinants generally associated with the antigen-binding
site of an antibody.
Antibodies of the Invention
The invention relates to an antibody that binds to human CD38 (SEQ
ID NO: 52), wherein the antibody does not bind to a variant of
human CD38 wherein Asp in position 202 has been substituted with
Gly to the same degree that it binds to human CD38. In one
embodiment, the EC50 of the binding of the antibody to the variant
of human CD38 wherein Asp in position 202 has been substituted with
Gly is less than 50%, such as less than 10%, less than 5%, or less
than 1% of the EC50 of the binding of the antibody to human
CD38.
In one embodiment,the antibody as defined above binds to a variant
of human CD38 wherein Gln in position 272 has been substituted with
Arg to the same degree that it binds to human CD38. In one
embodiment, the EC50 of the binding of the antibody to the variant
of human CD38 wherein Gln in position 272 has been substituted with
Arg is at least 80%, such as at least 90%, such as at least 95%,
such as at least 98% of the EC50 of the binding of the antibody to
human CD38.
In one embodiment,the antibody as defined in any of the embodiments
above binds to a variant of human CD38 wherein the Ser in position
274 has been substituted with Phe to the same degree that it binds
to human CD38. In one embodiment, the EC50 of the binding of the
antibody to a variant of human CD38 is at least 75%, such as at
least 80%, such as at least 90%, such as at least 95%, such as at
least 98% of the EC50 of the binding of the antibody to human
CD38.
In one embodiment,the antibody as defined above possesses the
following binding characteristics: (i) it does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human
CD38, (ii) it binds to a variant of human CD38 wherein Gln in
position 272 has been substituted with Arg to the same degree that
it binds to human CD38, (iii) it binds to a variant of human CD38
wherein the Ser in position 274 has been substituted with Phe to
the same degree that it binds to human CD38.
In one embodiment, the antibody as defined in any of the
embodiments above binds human CD38 and has an inhibitory effect on
the CD38 cyclase activity and a stimulatory effect on the CD38
hydrolase activity as measured in the assays of Example 8, such as
wherein the inhibitory effect is at least 50-66% compared to the
inhibitory effect on the CD38 cyclase activity in the absence of
antibody.
In one embodiment, the antibody as defined in any of the
embodiments above is encoded by a human heavy chain nucleic acid
comprising a nucleotide sequence in its variable region as set
forth in SEQ ID NO: 1, 6, 11, 16 or 21, and a human light chain
nucleic acid comprising a nucleotide sequence in its variable
region as set forth in SEQ ID NOs: 26, 31, 36, 41 or 46.
In one embodiment, the antibody as defined in any of the
embodiments above is encoded by a human heavy chain and a human
light chain nucleic acid comprising nucleotide sequences in their
variable regions as set forth in SEQ ID NOs: 1 and 26, 6 and 31, 11
and 36, 16 and 41, or 21 and 46, respectively.
In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH CDR3 comprising a) the sequence as
set forth in SEQ ID NOs: 5, 10, 15, 20 or 25, or b) a variant of
said sequence, such as a variant having at most 1, 2 or 3 amino
acid modifications, preferably substitutions, such as conservative
substitutions.
In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH CDR3 having the sequence set forth
in SEQ ID NOs: 5, 10, 15, 20 or 25, and comprising a VL CDR3 having
the sequence set forth in SEQ ID NO: 30, 35, 40, 45 or 50.
In one embodiment, the antibody as defined in any of the
embodiments above comprises SEQ ID NO: 5 and SEQ ID NO: 30, or SEQ
ID NO: 10 and SEQ ID NO: 35, or SEQ ID NO: 15 and SEQ ID NO: 40, or
SEQ ID NO: 20 and SEQ ID NO: 45, or SEQ ID NO: 25 and SEQ ID NO: 50
as the VH CDR3 and VL CDR3 respectively.
In one embodiment, the antibody as defined in any of the
embodiments above comprises (i) a VH CDR1 having the sequence as
set forth in any of the sequences SEQ ID NOs: 3, 8, 13, 18 and 23,
a VH CDR2 having the sequence as set forth in any of the sequences
SEQ ID NOs: 4, 9, 14, 19 and 24, a VH CDR3 having the sequence as
set forth in any of the sequences SEQ ID NOs: 5, 10, 15, 20 and 25,
a VL CDR1 having the sequence as set forth in any of the sequences
SEQ ID NO: 28, 33, 38, 43 and 48, a VL CDR2 having the sequence as
set forth in any of the sequences SEQ ID NOs: 29, 34, 39, 44 and
49, a VL CDR3 having the sequence as set forth in any of the
sequences SEQ ID NOs: 30, 35, 40, 45 and 50, (ii) a VH CDR1 having
the sequence as set forth in SEQ ID NO: 3, a VH CDR2 having the
sequence as set forth in SEQ ID NOs: 4, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 5, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 28, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 29, a VL CDR3 having the
sequence as set forth in SEQ ID NO: 30, iii) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 8, a VH CDR2 having the
sequence as set forth in SEQ ID NOs: 9, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 10, a VL CDR1 having the
sequence as set forth in SEQ ID NO 33, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 34, a VL CDR3 having the
sequence as set forth in SEQ ID NO: 35, (iv) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 13, a VH CDR2 having the
sequence as set forth in SEQ ID NO: 14, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 15, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 38, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 39, a VL CDR3 having the
sequence as set forth in SEQ ID NO: 40, (v) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 18, a VH CDR2 having the
sequence as set forth in SEQ ID NOs: 19, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 20, a VL CDR1 having the
sequence as set forth in SEQ ID NO: 43, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 44, a VL CDR3 having the
sequence as set forth in SEQ ID NO: 45, (vi) a VH CDR1 having the
sequence as set forth in SEQ ID NO: 23, a VH CDR2 having the
sequence as set forth in SEQ ID NOs: 24, a VH CDR3 having the
sequence as set forth in SEQ ID NO: 25, a VL CDR1 having the
sequence as set forth in SEQ ID NO 48, a VL CDR2 having the
sequence as set forth in SEQ ID NO: 49, a VL CDR3 having the
sequence as set forth in SEQ ID NO: 50, or (vii) a variant of any
of the antbodies defined above, wherein said variant preferably has
at most 1, 2 or 3 amino acid modifications, more preferably
amino-acid substitutions, such as conservative amino acid
substitutions in one or more of said sequences.
In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH region (i) comprising the sequence
of SEQ ID NOs: 2, 7, 12, 17 or 22, or (ii) having at least 80%
identity, such as 90%, or 95%, or 97%, or 98%, or 99% or 100%
identity to the VH region sequence set forth in SEQ ID NOs: 2, 7,
12, 17 or 22.
In one embodiment, the antibody as defined in any of the
embodiments above comprises a VL region (i) comprising the sequence
of SEQ ID NOs: 27, 32, 37, 42 or 47, or (ii) having at least 80%
identity, such as 90%, or 95%, or 97%, or 98%, or 99% or 100%
identity to a VL region sequence selected from the group consisting
of: SEQ ID NOs: 27, 32, 37, 42 or 47.
In one embodiment, the antibody as defined in any of the
embodiments above comprises a VH region comprising any of the
sequences of SEQ ID NOs: 2, 7, 12, 17 and 22, and a VL region
comprising any of the sequences of SEQ ID NOs: 27, 32, 37, 42 and
47.
In one embodiment, the antibody as defined in any of the
embodiments above comprises (i) a VH region comprising the sequence
as set forth in SEQ ID NO: 2, and a VL region comprising any the
sequence as set forth in SEQ ID NO: 27, (ii) a VH region comprising
the sequence as set forth in SEQ ID NO: 7, and a VL region
comprising any the sequence as set forth in SEQ ID NO: 32, (iii) a
VH region comprising the sequence as set forth in SEQ ID NO: 12,
and a VL region comprising any the sequence as set forth in SEQ ID
NO: 37, (iv) a VH region comprising the sequence as set forth in
SEQ ID NO: 17, and a VL region comprising any the sequence as set
forth in SEQ ID NO: 42, or (v) a VH region comprising the sequence
as set forth in SEQ ID NO: 22, and a VL region comprising any the
sequence as set forth in SEQ ID NO: 47.
In one embodiment, the invention relates to an anti-CD38 antibody
which binds to the same epitope on CD38 as an anti-CD38 antibody as
described in any one of the embodiments above.
In one embodiment, the invention relates to an anti-CD38 antibody
which has substantially the same specific binding characteristics
for binding human CD38 as described in any one of the embodiments
above.
In one embodiment, the antibody as defined in any of the
embodiments above is capable of inducing antibody-dependent
cellular cytotoxicity (ADCC), such as in Daudi cells, preferably
with an EC.sub.50 value of 5 nM or less, e.g. 1 nM or less, such as
0.2 nM or less, as determined by the method described in Example 6
herein.
In one embodiment, the antibody as defined in any of the
embodiments above is not capable of inducing ADCC in Daudi cells
according to the method described in Example 6 herein.
In one embodiment, the antibody as defined in any of the
embodiments above is not capable of inducing complement-dependent
cytotoxicity (CDC) in CHO-CD38 cells.
In one embodiment, the antibody as defined in any of the
embodiments above binds to human CD38 with a K.sub.D of 10.sup.-8 M
or less, preferably with a K.sub.D of 10.sup.-9 M or less.
In one embodiment, the antibody as defined in any of the
embodiments above is a human monovalent antibody.
In one embodiment, the antibody as defined in any of the
embodiments above is a full length IgG1, IgG2, IgG3, IgG4, IgD,
IgA, IgE, or IgM antibody, such as an IgG1 antibody, preferably an
IgG1,.kappa. antibody or an IgM antibody, preferably an IgM,.kappa.
antibody.
In one embodiment, the antibody as defined in any of the
embodiments above is an antibody fragment or a single-chain
antibody.
In one embodiment, the antibody as defined in any of the
embodiments above is an effector-function-deficient antibody, such
as a stabilized human IgG4 antibody.
In one embodiment, such stabilized IgG4 antibody is an antibody
wherein arginine at position 409 in the heavy chain constant region
of human IgG4 is substituted with lysine, threonine, methionine, or
leucine, preferably lysine. In one embodiment, such antibody
comprises a Lys residue at the position corresponding to 409 or the
CH3 region of the antibody has been replaced by the CH3 region of
human IgG1, of human IgG2 or of human IgG3. In one embodiment, such
antibody does not comprise a Cys-Pro-Pro-Cys sequence in the hinge
region. In another embodiment, such antibody does comprise a
Cys-Pro-Pro-Cys sequence in the hinge region.
In one embodiment, the antibody as defined in any of the
embodiments above is a monovalent antibody.
In one embodiment, such monovalent antibody is constructed by a
method comprising: i) providing a nucleic acid construct encoding
the light chain of said monovalent antibody, said construct
comprising a nucleotide sequence encoding the VL region of SEQ ID
NO: 27, 32, 37, 42 or 47 and a nucleotide sequence encoding the
constant CL region of an Ig, wherein said nucleotide sequence
encoding the VL region of a selected antigen specific antibody and
said nucleotide sequence encoding the CL region of an Ig are
operably linked together, and wherein, in case of an IgG1 subtype,
the nucleotide sequence encoding the CL region has been modified
such that the CL region does not contain any amino acids capable of
forming disulfide bonds or covalent bonds with other peptides
comprising an identical amino acid sequence of the CL region in the
presence of polyclonal human IgG or when administered to an animal
or human being; ii) providing a nucleic acid construct encoding the
heavy chain of said monovalent antibody, said construct comprising
a nucleotide sequence encoding the VH region of SEQ ID NO: 2, 7, 12
17 or 22 and a nucleotide sequence encoding a constant CH region of
a human Ig, wherein the nucleotide sequence encoding the CH region
has been modified such that the region corresponding to the hinge
region and, as required by the Ig subtype, other regions of the CH
region, such as the CH3 region, does not comprise any amino acid
residues which participate in the formation of disulphide bonds or
covalent or stable non-covalent inter-heavy chain bonds with other
peptides comprising an identical amino acid sequence of the CH
region of the human Ig in the presence of polyclonal human IgG or
when administered to an animal human being, wherein said nucleotide
sequence encoding the VH region of a selected antigen specific
antibody and said nucleotide sequence encoding the CH region of
said Ig are operably linked together; iii) providing a cell
expression system for producing said monovalent antibody; iv)
producing said monovalent antibody by co-expressing the nucleic
acid constructs of (i) and (ii) in cells of the cell expression
system of (iii).
In one embodiment, the C.sub.H region comprising the C.sub.H2 and
C.sub.H3 regions has been modified such that the region
corresponding to the hinge region and, if the immunoglobulin is not
an IgG4 subtype, other regions of the C.sub.H region, such as the
C.sub.H3 region, do not comprise any amino acid residues, which are
capable of forming disulfide bonds with an identical C.sub.H region
or other covalent or stable non-covalent inter-heavy chain bonds
with an identical C.sub.H region in the presence of polyclonal
human IgG.
In one embodiment, such monovalent antibody is of the IgG4 subtype,
but the C.sub.H3 region has been modified so that one or more of
the following amino acid substitutions have been made: Thr (T) in
position 366 has been replaced by Ala (A); Leu (L) in position 368
has been replaced by Ala (A); Leu (L) in position 368 has been
replaced by Val (V); Phe (F) in position 405 has been replaced by
Ala (A); Phe (F) in position 405 has been replaced by Leu (L); Tyr
(Y) in position 407 has been replaced by Ala (A); Arg (R) in
position 409 has been replaced by Ala (A).
In one embodiment, the heavy chain of such monovalent antibody has
been modified such that the entire hinge has been deleted.
In one embodiment, the sequence of said monovalent antibody has
been modified so that it does not comprise any acceptor sites for
N-linked glycosylation.
In one embodiment, the antibody as defined in any of the
embodiments above inhibits the CD38 catalyzed synthesis of cGDPR by
at least 25%, such as at least 30% after 90 minutes at a
concentration of 3 .mu.g/ml as determined by the spectophotometric
method described in Example 8 of the specification.
In one embodiment, the antibody as defined in any of the
embodiments above inhibits the CD38 catalyzed synthesis of cADPR by
at least 25%, such as at least 30% after 90 minutes at a
concentration of 3 .mu.g/ml as determined by the HPLC method
described in Munshi et al., J. Biol. Chem. 275, 21566-21571
(2000).
In one embodiment, the antibody stimulates the hydrolase activity
of CD38 by at least 25%.
In one embodiment, the antibody stimulates the NAD hydrolase
activity of CD38 by at least 25%.
In one embodiment, the antibody as defined in any of the
embodiments above stimulates the cADPR-hydrolase activity of CD38
by at least 25%.
In one embodiment, the antibody as defined in any of the
embodiments above inhibits the ability of CD38 to catalyze the
formation, via a base-exchange reaction, of NAADP with an IC50 of
below 0.5 .mu.g/mL, such as of below 0.2 .mu.g/mL by the method
described in Example 8 of the specification.
In one embodiment, the invention relates to an antibody drug
conjugate comprising an antibody as defined in any of the
embodiments above, wherein the antibody has been conjugated to a
cytotoxic agent, a radioisotope, or a drug. In one embodiment, the
antibody has been conjugated to an auristatin or a functional
peptide analog or derivate thereof via a linker.
In one embodiment, the invention relates to a bispecific antibody
comprising an antibody as defined in any of the embodiments above
and a second binding specificity for a human effector cell or a
cancer antigen. In one embodiment, the second binding specificity
is for a human Fc receptor or for a T cell receptor, such as
CD3.
In one embodiment, the invention relates to an isolated nucleic
acid encoding an antibody as defined in any of the embodiments
above.
In one embodiment, the invention relates to an expression vector
comprising a nucleotide sequence encoding one or more of the amino
acid sequences as defined in any of the embodiments above.
In one embodiment, the expression vector further comprises a
nucleotide sequence encoding the constant region of a light chain,
a heavy chain or both light and heavy chains of a human
antibody.
In one embodiment, the invention relates to a recombinant
eukaryotic or prokaryotic host cell which produces an antibody as
defined in any of the embodiments above.
In one embodiment, the invention relates to a pharmaceutical
composition comprising an antibody, an immunoconjugate, a
bispecific antibody, or an expression vector as defined in any of
the embodiments above and a pharmaceutically acceptable
carrier.
In one embodiment, the invention relates to an antibody as defined
in any of the embodiments above for use as a medicament.
In one embodiment, the invention relates to an antibody as defined
in any of the embodiments above for use in inhibiting growth and/or
proliferation, migration or inducing phagocytosis of a tumor cell
expressing CD38.
In one embodiment, the invention relates to an antibody as defined
in any of the embodiments above for use in treating rheumatoid
arthritis.
In one embodiment, the invention relates to an antibody as defined
in any of the embodiments above for use in treating a disorder
selected from chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (adults)
(AML), mantle cell lymphoma, follicular lymphoma, and diffuse large
B-cell lymphoma.
In one embodiment, the invention relates to an antibody as defined
in any of the embodiments above for use in treating multiple
myeloma.
In one embodiment, the invention relates to a method for producing
an anti-CD38 antibody as defined in any of the embodiments above,
said method comprising the steps of a) culturing a host cell as
defined in any of the embodiments above, and b) purifying the
anti-CD38 antibody from the culture media.
In one embodiment, the invention relates to diagnostic composition
comprising an antibody as defined in any of the embodiments
above.
In one embodiment, the invention relates to a method for detecting
the presence of CD38 antigen, or a cell expressing CD38, in a
sample comprising: contacting the sample with an anti-CD38 antibody
as defined in any of the embodiments above under conditions that
allow for formation of a complex between the antibody or bispecific
molecules and CD38; and analyzing whether a complex has been
formed.
In one embodiment, the invention relates to a kit for detecting the
presence of CD38 antigen, or a cell expressing CD38, in a sample
comprising an anti-CD38 antibody as defined in any of the
embodiments above and instructions for use of the kit.
In one embodiment, the invention relates to an anti-idiotypic
antibody which binds to an anti-CD38 antibody as defined in any of
the embodiments above.
In one embodiment, the invention relates to a method of inhibiting
growth and/or proliferation migration or inducing phagocytosis of a
cell expressing CD38, comprising administration of an antibody, an
immunoconjugate, a bispecific antibody, an expression vector or a
pharmaceutical composition as defined in any of the embodiments
above, such that the growth and/or proliferation, migration or
phagocytosis of the cell is inhibited.
In one embodiment, the invention relates to a method of treating a
disease or disorder involving cells expressing CD38 in a subject,
which method administration of an antibody, an immunoconjugate, a
bispecific antibody, an expression vector or a pharmaceutical
composition as defined in any of the embodiments above to a subject
in need thereof.
In one embodiment, the disease or disorder is rheumatoid
arthritis.
In another embodiment, the disease or disorder is selected from
chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia
(ALL), acute myelogenous leukemia (adults) (AML), mantle cell
lymphoma, follicular lymphoma, and diffuse large B-cell
lymphoma.
In yet another embodiment, the disease or disorder is multiple
myeloma.
In one embodiment, the method as defined in any of the embodiments
above comprises administration of one or more further therapeutic
agents to the subject, such as one or more further therapeutic
agents are selected from a chemotherapeutic agent, an
anti-inflammatory agent, or an immunosuppressive and/or
immunomodulatory agent. In one embodiment, the one or more further
therapeutic agents are selected from a group consisting of
cisplatin, gefitinib, cetuximab, rituximab, ofatumumab,
bevacizumab, erlotinib, bortezomib, thalidomide, pamidronate,
zoledronic acid, clodronate, risendronate, ibandronate, etidronate,
alendronate, tiludronate, arsenic trioxide, lenalidomide,
dexamethasone, prednisolone, filgrastim, pegfilgrastim,
sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.
An embodiment of the invention provides an antibody that binds to
human CD38, wherein the antibody does not bind to a variant of
human CD38 wherein Asp in position 202 has been substituted with
Gly.
An embodiment of the invention provides an antibody according to
the embodiment above, wherein the EC50 of the binding of the
antibody to a variant of human CD38 is less than 50%, such as less
than 10%, less than 5%, or less than 1% of the EC50 of the binding
of the peptide to human CD38.
An embodiment of the invention provides an antibody according to
any of the above embodiments, wherein the antibody binds to a
variant of human CD38 wherein the Gln in position 272 has been
substituted with Arg to the same degree that it binds to human
CD38.
An embodiment of the invention provides an antibody according to
the above embodiment, wherein the EC50 of the binding of the
antibody to a variant of human CD38 is at least 80%, such as at
least 90%, such as at least 95%, such as at least 98% of the EC50
of the binding of the peptide to human CD38.
An embodiment of the invention provides an antibody according to
any of the above embodiments, wherein the antibody binds to a
variant of human CD38 wherein the Ser in position 274 has been
substituted with Phe to the same degree that it binds to human
CD38.
An embodiment of the invention provides an antibody according to
the above embodiment, wherein the EC50 of the binding of the
antibody to a variant of human CD38 is at least 75%, such as at
least 80%, such as at least 90%, such as at least 95%, such as at
least 98% of the EC50 of the binding of the peptide to human
CD38.
An embodiment of the invention provides an antibody according to
any of the above embodiments,wherein the antibody possesses the
following binding characteristics: (i) it does not bind to a
variant of human CD38 wherein Asp in position 202 has been
substituted with Gly to the same degree that it binds to human CD38
(ii) it binds to a variant of human CD38 wherein the Gln in
position 272 has been substituted with Arg to the same degree that
it binds to human CD38 (iii) it binds to a variant of human CD38
wherein the Ser in position 274 has been substituted with Phe to
the same degree that it binds to human CD38.
An embodiment of the invention provides an antibody according to
any of the above embodiments, that binds human CD38 and has an
inhibitory effect on the CD38 cyclase activity and a stimulatory
effect on the CD38 hydrolase acitivity as measured in the assays of
Example 8.
An embodiment of the invention provides an antibody according to
the above embodiment, wherein the inhibitory effect is at least
50-66% compared to CD38 alone.
An embodiment of the invention provides an antibody binding to
human CD38 encoded by a human heavy chain nucleic acids comprising
nucleotide sequences in their variable regions as set forth the in
seq id no.: 1, 6, 11, 16 or 21, and a human light chain comprising
nucleotide sequences in their variable regions as set forth in seq
id no. 26, 31, 36, 41 or 46, and comprising conservative sequence
modifications of the sequences set forth above.
An embodiment of the invention provides an antibody according to
the above embodiment, encoded by a human heavy chain and a human
light chain nucleic acids comprising nucleotide sequences in their
variable regions as set forth the in seq id no.: 1 and 26, 6 and
31, 11 and 36, 16 and 41 or 21 and 46, respectively, and comprising
conservative sequence modifications of the sequences set forth
above.
An embodiment of the invention provides an antibody binding to
human CD38 comprising a VH CDR3 region having a) the sequence as
set forth in SEQ ID NOs: 5, 10, 15, 20 or 25 30 or b) a variant of
said sequence, such as a variant having at most 1,2 or 3 amino-acid
modifications, preferably substitutions, such as conservative
substitutions.
An embodiment of the invention provides an antibody binding to
human CD38 comprising a VH CDR3 region having the sequence as set
forth in SEQ ID NOs: 5, 10, 15, 20, 25 or 30 and comprising a VL
CDR3 region having the sequence set forth in SEQ ID NO: 30, 35, 40,
45 or 50;
An embodiment of the invention provides an antibody binding to
human CD38 comprising a VH CDR3 region having the sequence as set
forth in SEQ ID NO: 5 and a VL CDR3 region comprising SEQ ID NO:
30, or SEQ ID NO: 10 and SEQ ID NO: 35, or SEQ ID NO: 15 and SEQ ID
NO: 40, or SEQ ID NO: 20 and SEQ ID NO: 45, or SEQ ID NO: 25 and
SEQ ID NO: 45, or SEQ ID NO: 30 and SEQ ID NO: 50, as VH CDR3
region and VL CDR3 region respectively.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody
comprises a VH CDR1 region having the sequence as set forth in any
of the sequences SEQ ID NOs: 3, 8, 13, 18 or 23, a VH CDR2 region
having the sequence as set forth in any of the sequences SEQ ID
NOs: 4, 9, 14, 19 or 24, a VL CDR3 region having the sequence as
set forth in any of the seqeuences SEQ ID NOs: 30, 35, 40, 45 or
50, and a VH CDR3 region having the sequence as set forth in SEQ ID
NOs: 5, 10, 15, 20 or 25.
An embodiment of the invention provides an antibody which binds to
CD38, wherein the antibody comprises a VH CDR1 region having the
sequence as set forth in any of the sequences SEQ ID NOs: 3, 8, 13,
18 or 23, a VH CDR2 region having the sequence as set forth in any
of the sequences SEQ ID NOs: 4, 9, 14, 19 or 24, a VH CDR3 region
having the sequence as set forth in SEQ ID NOs: 5, 10, 15, 20 or
25, a VL CDR1 region as set forth in SEQ ID NOs: 28, 33, 38, 43 or
48, a VL CDR2 region as set forth in SEQ ID NOs: 29, 34, 39, 44 or
49, a VL CDR3 region having the sequence as set forth in any of the
sequences SEQ ID NOs: 30, 35, 40, 45 or 50 or
a variant of said antibody, wherein said variant preferably has at
most 1,2 or 3 amino-acid modifications, more preferably amino-acid
substitutions, such as conservative amino-acid substitutions in
said sequences.
An embodiment of the invention provides an antibody which binds to
CD38, wherein the antibody comprises a VH CDR1 region having the
sequence as set forth in any of the sequences SEQ ID NOs: 3, 8, 13,
18 or 23, a VH CDR2 region having the sequence as set forth in any
of the sequences SEQ ID NOs: 4, 9, 14, 19 or 24, a VH CDR3 region
having the sequence as set forth in SEQ ID NOs: 5, 10, 15, 20 or
25, a VL CDR1 region as set forth in SEQ ID NOs: 28, 33, 38, 43 or
48, a VL CDR2 region as set forth in SEQ ID NOs: 29, 34, 39, 44 or
49, a VL CDR3 region having the sequence as set forth in any of the
sequences SEQ ID NOs: 30, 35, 40, 45 or 50;
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, comprising a VH having
at least 80% identity, such as 90%, or 95%, or 97%, or 98%, or 99%
or 100% identity to the VH region sequence set forth in SEQ ID NOs:
2, 7, 12, 17, or 22.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, comprising a VL having
at least 80% identity, such as 90%, or 95%, or 97%, or 98%, or 99%
or 100% identity to a VL region sequence selected from the group
consisting of: SEQ ID NOs: 27, 32, 37, 42 or 47.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, comprising a VH region
comprising the sequence of SEQ ID NOs: 2, 7, 12, 17 or 22 and a VL
region comprising the sequence of SEQ ID NOs: SEQ ID NOs: 27, 32,
37, 42 or 47.
according to any of the above embodimentsaccording to any of the
above embodimentsaccording to any of the above embodiments
An embodiment of the invention provides an antibody which competes
with an antibody according to any of the above embodiments,for
binding to CD38.
An embodiment of the invention provides an anti-CD38 antibody,
which competes for CD38 binding with an anti-CD38 antibody
comprising a VH region comprising any of the sequences of SEQ ID
NOs: 2, 7, 12, 17 or 22 and a VL region comprising any of the
sequences of SEQ ID NO: 27, 32, 37, 42 or 47.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody
binds to the same epitope on CD38 as an anti-CD38 antibody as
described in any of the above embodiments.
An embodiment of the invention provides an antibody having
substantially the same specific binding characteristics for binding
human CD38 has an antibody according to any of the above
embodiments.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
capable of inducing complement-dependent cytotoxicity (CDC) in
CHO-CD38 cells.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
capable of inducing antibody-dependent cellular cytotoxicity
(ADCC).
An embodiment of the invention provides an anti-CD38 antibody of
claim 25, wherein said antibody induces ADCC in Daudi cells,
preferably with an EC.sub.50 value of 5 nM or less, e.g. 1 nM or
less, such as 0.2 nM or less, as determined by the method described
in Example 6 herein.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
not capable of inducing ADCC.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
not capable of inducing complement-dependent cytotoxicity
(CDC).
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments,wherein the antibody
binds to human CD38 with a K.sub.D of 10.sup.-8 M or less,
preferably with a K.sub.D of 10.sup.-9 M or less.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments,wherein the antibody
comprises: a heavy chain variable region derived from a human
germline V.sub.H sequence selected from the group consisting of:
IGHV1-69*04, and/or IGHJ3*02 a light chain variable region derived
from a human germline V.kappa. sequence selected from the group
consisting of: IGKV1D-16*01, and/or IGKJ4*01.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, which is a human
antibody.
An embodiment of the invention provides an antibody according to
any of the above embodiments, characterized in that it is a full
length IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody, such
as an IgG1 antibody, preferably an IgG1, .kappa. antibody or an IgM
antibody, preferably an IgM,.kappa. antibody.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
an antibody fragment or a single-chain antibody.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
conjugated to another moiety, such as a cytotoxic moiety, a
radioisotope or a drug.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
an effector-function-deficient antibody.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiment, wherein the effector-function-deficient
anti-CD38 antibody is a stabilized human IgG4 antibody.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiment, wherein the stabilized IgG4 antibody is an
antibody wherein arginine at position 409 in the heavy chain
constant region of human IgG4 is substituted with lysine,
threonine, methionine, or leucine, preferably lysine.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiment, wherein said antibody comprises a Lys residue
at the position corresponding to 409 or the CH3 region of the
antibody has been replaced by the CH3 region of human IgG1, of
human IgG2 or of human IgG3.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiments, wherein said antibody does not comprise a
Cys-Pro-Pro-Cys sequence in the hinge region.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiments, wherein said antibody does comprise a
Cys-Pro-Pro-Cys sequence in the hinge region.
An embodiment of the invention provides an anti-CD38 antibody
according to any of the above embodiments, wherein the antibody is
a monovalent antibody.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiment, wherein said monovalent antibody is
constructed by a method comprising: i) providing a nucleic acid
construct encoding the light chain of said monovalent antibody,
said construct comprising a nucleotide sequence encoding the VL
region of a selected antigen specific antibody and a nucleotide
sequence encoding the constant CL region of an Ig, wherein said
nucleotide sequence encoding the VL region of a selected antigen
specific antibody and said nucleotide sequence encoding the CL
region of an Ig are operably linked together, and wherein, in case
of an IgG1 subtype, the nucleotide sequence encoding the CL region
has been modified such that the CL region does not contain any
amino acids capable of forming disulfide bonds or covalent bonds
with other peptides comprising an identical amino acid sequence of
the CL region in the presence of polyclonal human IgG or when
administered to an animal or human being; ii) providing a nucleic
acid construct encoding the heavy chain of said monovalent
antibody, said construct comprising a nucleotide sequence encoding
the VH region of a selected antigen specific antibody and a
nucleotide sequence encoding a constant CH region of a human Ig,
wherein the nucleotide sequence encoding the CH region has been
modified such that the region corresponding to the hinge region
and, as required by the Ig subtype, other regions of the CH region,
such as the CH3 region, does not comprise any amino acid residues
which participate in the formation of disulphide bonds or covalent
or stable non-covalent inter-heavy chain bonds with other peptides
comprising an identical amino acid sequence of the CH region of the
human Ig in the presence of polyclonal human IgG or when
administered to an animal human being, wherein said nucleotide
sequence encoding the VH region of a selected antigen specific
antibody and said nucleotide sequence encoding the CH region of
said Ig are operably linked together; iii) providing a cell
expression system for producing said monovalent antibody; iv)
producing said monovalent antibody by co-expressing the nucleic
acid constructs of (i) and (ii) in cells of the cell expression
system of (iii).
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiment, wherein the monovalent antibody comprises (i)
a variable region of an antibody according to any of the above
embodiments,or an antigen binding part of the said region, and (ii)
a C.sub.H region of an immunoglobulin or a fragment thereof
comprising the C.sub.H2 and C.sub.H3 regions, wherein the C.sub.H
region or fragment thereof has been modified such that the region
corresponding to the hinge region and, if the immunoglobulin is not
an IgG4 subtype, other regions of the C.sub.H region, such as the
C.sub.H3 region, do not comprise any amino acid residues, which are
capable of forming disulfide bonds with an identical C.sub.H region
or other covalent or stable non-covalent inter-heavy chain bonds
with an identical C.sub.H region in the presence of polyclonal
human IgG.
An embodiment of the invention provides an anti-CD38 antibody of
the above embodiments wherein said monovalent antibody is of the
IgG4 subtype, but the C.sub.H3 region has been modified so that one
or more of the following amino acid substitutions have been made:
Thr (T) in position 366 has been replaced by Ala (A); Leu (L) in
position 368 has been replaced by Ala (A); Leu (L) in position 368
has been replaced by Val (V); Phe (F) in position 405 has been
replaced by Ala (A); Phe (F) in position 405 has been replaced by
Leu (L); Tyr (Y) in position 407 has been replaced by Ala (A); Arg
(R) in position 409 has been replaced by Ala (A).
An embodiment of the invention provides an anti-CD38 antibody of
any of the above embodiments, the heavy chain has been modified
such that the entire hinge has been deleted.
An embodiment of the invention provides an anti-CD38 antibody of
any of the above embodiments, wherein the sequence of said
monovalent antibody has been modified so that it does not comprise
any acceptor sites for N-linked glycosylation.
An embodiment of the invention provides an antibody according to
any of the above embodiments, which inhibits the synthesis of cGDPR
by at least 25%, such as at least 30% after 90 minutes as
determined by spectophotometric method described in Example 8 of
the specification.
An embodiment of the invention provides an antibody according to
any of the above embodiments wich inhibits the synthesis of cADPR
by at least 25%, such as at least 30% after 90 minutes at a
concentration of 3 .mu.g/ml as determined by the HPLC method
described in Munshi et al., J. Biol. Chem. 275, 21566-21571
(2000).
An embodiment of the invention provides an antibody according to
any of the above embodiments, which stimulate the hydrolase
activity of CD38 by at least 25%.
An embodiment of the invention provides an antibody according to
any of the above embodiments, which stimulate the NAD hydrolase
activity by at least 25%.
An embodiment of the invention provides an antibody according to
any of the above embodiments, which stimulate the cADPR-hydrolase
activity by at least 25%.
An embodiment of the invention provides an isolated nucleic acid
encoding a peptide according to the any of the above
embodiments.
An embodiment of the invention provides an expression vector
comprising a nucleotide sequence encoding one or more of the amino
acid sequences according to any of the above embodiments.
An embodiment of the invention provides an expression vector
according to the above embodiment, further comprising a nucleotide
sequence encoding the constant region of a light chain, a heavy
chain or both light and heavy chains of a human antibody.
An embodiment of the invention provides a recombinant eukaryotic or
prokaryotic host cell which produces an antibody as defined in any
of the above embodiments.
An embodiment of the invention provides a hybridoma which produces
an antibody as defined in any of the above embodiments.
An embodiment of the invention provides a pharmaceutical
composition comprising an antibody as defined in any of the above
embodiments, and a pharmaceutically acceptable carrier.
An embodiment of the invention provides an antibody as defined in
any of the embodiments above for use as a medicament.
An embodiment of the invention provides an antibody as defined in
any of the embodiments above for use in inhibiting growth and/or
proliferation, migration or inducing phagocytosis of a tumor cell
expressing CD38.
An embodiment of the invention provides an antibody as defined in
any of the embodiments above for use in treating rheumatoid
arthritis.
An embodiment of the invention provides an antibody as defined in
any of the embodiments above for use in treating multiple
myeloma.
An embodiment of the invention provides an antibody as defined in
any of the embodiments above for use in treating multiple
sclerosis.
An embodiment of the invention provides an antibody as defined in
any of the embodiments above for use in treating B-cell neoplasms
such as any one of the following: small lymphocytic lymphoma,
B-cell prolymphocytic lymphoma, B-cell chronic lymphocytic
leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular
lymphoma, diffuse large B-cell lymphoma, multiple myeloma,
lymphoplasmacytic lymphoma, splenic margina zone lymphoma, plasma
cell neoplasms, such as plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition disease, heavy chain disease,
MALT lymphoma, nodal marginal B cell lymphoma, intravascular large
B cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy
cell leukemia, primary effusion lymphoma or AIDS-related
non-Hodgkins lymphoma
An embodiment of the invention provides a method for inhibiting
growth and/or proliferation, migration or inducing phagocytosis of
a tumor cell expressing CD38, comprising administration, to an
individual in need thereof, of an antibody of any of the above
embodiments.
An embodiment of the invention provides a method for producing an
anti-CD38 antibody of any of the above embodiments, said method
comprising the steps of a) culturing a host cell of claim 52 or a
hybridoma of the above embodiment, and b) purifying the anti-CD38
antibody from the culture media.
An embodiment of the invention provides a diagnostic composition
comprising an antibody as defined in any of the above
embodiments.
An embodiment of the invention provides a method for detecting the
presence of CD38 antigen, or a cell expressing CD38, in a sample
comprising: contacting the sample with an anti-CD38 antibody of any
of the above embodiments under conditions that allow for formation
of a complex between the antibody or bispecific molecules and CD38;
and analyzing whether a complex has been formed.
An embodiment of the invention provides a kit for detecting the
presence of CD38 antigen, or a cell expressing CD38, in a sample
comprising an anti-CD38 antibody of any of the above embodiments or
and instructions for use of the kit.
An embodiment of the invention provides an anti-idiotypic antibody
which binds to an anti-CD38 antibody of any of the above
embodiments.
An embodiment of the invention provides a method of inhibiting
growth and/or proliferation of a cell expressing CD38, comprising
administration of a peptide according to any of the above
embodiments, an immunoconjugate according to the above embodiment,
a pharmaceutical composition according to the above embodiments or
an expression vector mentioned in the above embodiments, such that
the growth and/or proliferation, migration or phagocytosis of the
cell is inhibited.
An embodiment of the invention provides a method of treating a
disease or disorder involving cells expressing CD38 in a subject,
which method comprises administration of a peptide according to any
of the above embodiments, an immunoconjugate according to an
embodiment above, a pharmaceutical composition according to an
embodiment above, or an expression vector according to any one the
embodiments above to a subject in need thereof.
An embodiment of the invention provides a method of preventing a
disease or disorder involving cells expressing CD38 in a subject,
which method comprises administration of a peptide according to any
of the above embodiments, an immunoconjugate according to an
embodiment above, a pharmaceutical composition according to an
embodiment above, or an expression vector according to any one the
embodiments above to a subject in need thereof.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is rheumatoid
arthritis.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is B-cell
neoplasms such as any one of the following: small lymphocytic
lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic
lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma,
follicular lymphoma, diffuse large B-cell lymphoma, multiple
myeloma, lymphoplasmacytic lymphoma, splenic margina zone lymphoma,
plasma cell neoplasms, such as plasma cell myeloma, plasmacytoma,
monoclonal immunoglobulin deposition disease, heavy chain disease,
MALT lymphoma, nodal marginal B cell lymphoma, intravascular large
B cell lymphoma, primary effusion lymphoma, lymphomatoid
granulomatosis, non-Hodgkins lymphoma, Hodgkins lymphoma, hairy
cell leukemia, primary effusion lymphoma and AIDS-related
non-Hodgkins lymphoma.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is multiple
myeloma
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is autoimmune
disease.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is diabetes.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is multiple
sclerosis.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is Grave's
disease.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is
neuroinflammation.
An embodiment of the invention provides a method according to the
above embodiments wherein the disease or disorder is inflammation
of airway smooth muscle cells during asthma.
An embodiment of the invention provides a method according to the
above embodiments, wherein the method comprises administration of
one or more further therapeutic agents to the subject.
An embodiment of the invention provides a method according to the
above embodiment, wherein the one or more further therapeutic
agents are selected from a chemotherapeutic agent, an
anti-inflammatory agent, or an immunosuppressive and/or
immunomodulatory agent.
An embodiment of the invention provides a method according to the
above embodiment, wherein the one or more further therapeutic
agents are selected from a group consisting of cisplatin,
gefitinib, cetuximab, rituximab, bevacizumab, erlotinib,
bortezomib, thalidomide, pamidronate, zoledronic acid, clodronate,
risendronate, ibandronate, etidronate, alendronate, tiludronate,
arsenic trioxide, lenalidomide, filgrastim, pegfilgrastim,
sargramostim, suberoylanilide hydroxamic acid, and SCIO-469.
Monoclonal antibodies of the present invention may e.g. be produced
by the hybridoma method first described by Kohler et al., Nature
256, 495 (1975), or may be produced by recombinant DNA methods.
Monoclonal antibodies may also be isolated from phage antibody
libraries using the techniques described in, for example, Clackson
et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol.
222, 581-597 (1991). Monoclonal antibodies may be obtained from any
suitable source. Thus, for example, monoclonal antibodies may be
obtained from hybridomas prepared from murine splenic B cells
obtained from mice immunized with an antigen of interest, for
instance in form of cells expressing the antigen on the surface, or
a nucleic acid encoding an antigen of interest.
In one embodiment, the antibody of the invention is a human
antibody. Human monoclonal antibodies directed against CD38 may be
generated using transgenic or transchromosomal mice carrying parts
of the human immune system rather than the mouse system. Such
transgenic and transchromosomic mice include mice referred to
herein as HuMAb mice and KM mice, respectively, and are
collectively referred to herein as "transgenic mice".
The HuMAb mouse contains a human immunoglobulin gene miniloci that
encodes unrearranged human heavy (.mu. and .gamma.) and .kappa.
light chain immunoglobulin sequences, together with targeted
mutations that inactivate the endogenous .mu. and .kappa. chain
loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly,
the mice exhibit reduced expression of mouse IgM or .kappa. and in
response to immunization, the introduced human heavy and light
chain transgenes, undergo class switching and somatic mutation to
generate high affinity human IgG,.kappa. monoclonal antibodies
(Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. Handbook
of Experimental Pharmacology 113, 49-101 (1994) , Lonberg, N. and
Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding,
F. and Lonberg, N. Ann. N.Y. Acad. Sci 764 536-546 (1995)). The
preparation of HuMAb mice is described in detail in Taylor, L. et
al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al.,
International Immunology 5, 647-656 (1993), Tuaillon et al., J.
Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International
Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature
Biotechnology 14, 845-851 (1996). See also U.S. Pat. Nos.
5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397,
5,661,016, 5,814,318, 5,874,299, 5,770,429, 5,545,807, WO 98/24884,
WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO
01/09187.
The HCo7 mice have a JKD disruption in their endogenous light chain
(kappa) genes (as described in Chen et al., EMBO J. 12, 821-830
(1993)), a CMD disruption in their endogenous heavy chain genes (as
described in Example 1 of WO 01/14424), a KCo5 human kappa light
chain transgene (as described in Fishwild et al., Nature
Biotechnology 14, 845-851 (1996)), and a HCo7 human heavy chain
transgene (as described in U.S. Pat. No. 5,770,429).
The HCo12 mice have a JKD disruption in their endogenous light
chain (kappa) genes (as described in Chen et al., EMBO J. 12,
821-830 (1993)), a CMD disruption in their endogenous heavy chain
genes (as described in Example 1 of WO 01/14424), a KCo5 human
kappa light chain transgene (as described in Fishwild et al.,
Nature Biotechnology 14, 845-851 (1996)), and a HCo12 human heavy
chain transgene (as described in Example 2 of WO 01/14424).
In the KM mouse strain, the endogenous mouse kappa light chain gene
has been homozygously disrupted as described in Chen et al., EMBO
J. 12, 811-820 (1993) and the endogenous mouse heavy chain gene has
been homozygously disrupted as described in Example 1 of WO
01/09187. This mouse strain carries a human kappa light chain
transgene, KCo5, as described in Fishwild et al., Nature
Biotechnology 14, 845-851 (1996). This mouse strain also carries a
human heavy chain transchromosome composed of chromosome 14
fragment hCF (SC20) as described in WO 02/43478.
Splenocytes from these transgenic mice may be used to generate
hybridomas that secrete human monoclonal antibodies according to
well known techniques.
Human monoclonal or polyclonal antibodies of the present invention,
or antibodies of the present invention originating from other
species may also be generated transgenically through the generation
of another non-human mammal or plant that is transgenic for the
immunoglobulin heavy and light chain sequences of interest and
production of the antibody in a recoverable form therefrom. In
connection with the transgenic production in mammals, antibodies
may be produced in, and recovered from, the milk of goats, cows, or
other mammals. See for instance U.S. Pat. Nos. 5,827,690,
5,756,687, 5,750,172 and 5,741,957.
Further, human antibodies of the present invention or antibodies of
the present invention from other species may be generated and
identified through display-type technologies, including, without
limitation, phage display, retroviral display, ribosomal display,
and other techniques, using techniques well known in the art and
the resulting molecules may be subjected to additional maturation,
such as affinity maturation, as such techniques are well known in
the art (see for instance Hoogenboom et al., J. Mol. Biol. 227, 381
(1991) (phage display), Vaughan et al., Nature Biotech 14, 309
(1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942
(1997) (ribosomal display), Parmley and Smith, Gene 73, 305-318
(1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et
al., PNAS USA 87, 6378-6382 (1990), Russel et al., Nucl. Acids
Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews
130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84
(1992), and U.S. Pat. No. 5,733,743). If display technologies are
utilized to produce antibodies that are not human, such antibodies
may be humanized.
Competition for binding to CD38 or a portion of CD38 by two or more
anti-CD38 antibodies may be determined by any suitable technique.
Competition in the context of the present invention refers to any
detectably significant reduction in the propensity for a particular
molecule to bind a particular binding partner in the presence of
another molecule that binds the binding partner. Typically,
competition means an at least about 10% reduction, such as an at
least about 15%, or an at least about 20% reduction in binding
between an anti-CD38 antibody and (a) a form of CD38 (e.g.
"processed", "mature", "unprocessed", "not processed" or "immature"
CD38); (b) a form of free CD38 (e.g., a CD38 fragment produced by
in vivo processing); (c) a heterodimeric peptide composed of
another peptide associated with CD38, such as CD31 associated with
CD38; (d) a complex of CD38 and one or more substrates, such as
cAMP, NAD+ and/or cADPR; (e) a dimerized, associated and/or
processed dimer of CD38 with a soluble ligand, such as CD31; or (f)
a portion of CD38, caused by the presence of another anti-CD38
antibody as determined by, e.g., ELISA analysis or FACS analysis
(as described in the examples section) using sufficient amounts of
the two or more competing anti-CD38 antibodies and CD38 molecule.
It may also be the case that competition may exist between
anti-CD38 antibodies with respect to more than one form of CD38,
and/or a portion of CD38, e.g. in a context where the
antibody-binding properties of a particular region of CD38 are
retained in fragments thereof, such as in the case of a
well-presented linear epitope located in various tested fragments
or a conformational epitope that is presented in sufficiently large
CD38 fragments as well as in CD38.
Assessing competition typically involves an evaluation of relative
inhibitory binding using a first amount of a first molecule; a
second amount of a second molecule; and a third amount of a third
molecule (or a standard determined by binding studies that may be
reasonably compared to new binding data with respect to the first
and second molecules as a surrogate for actual contemporaneous
data), wherein the first, second, and third amounts all are
sufficient to make a comparison that imparts information about the
selectivity and/or specificity of the molecules at issue with
respect to the other present molecules. The first, second, and
third amounts may vary with the nature of the anti-CD38 antibody
and potential targets therefore at issue. For instance, for ELISA
assessments, similar to those described in the Examples section,
about 5-50 .mu.g (e.g., about 10-50 .mu.g, about 20-50 .mu.g, about
5-20 .mu.g, about 10-20 .mu.g, etc.) of anti-CD38 antibody and/or
CD38 targets are required to assess whether competition exists.
Conditions also should be suitable for binding. Typically,
physiological or near-physiological conditions (e.g., temperatures
of about 20-40.degree. C., pH of about 7-8, etc.) are suitable for
anti-CD38 antibody:CD38 binding. Often competition is marked by a
significantly greater relative inhibition than about 5% as
determined by ELISA and/or FACS analysis. It may be desirable to
set a higher threshold of relative inhibition as a
criteria/determinant of what is a suitable level of competition in
a particular context (e.g., where the competition analysis is used
to select or screen for new antibodies designed with the intended
function of blocking the binding of another peptide or molecule
binding to CD38 (e.g., the natural binding partners of CD38 such as
CD31, also called CD31 antigen, EndoCAM, GPIIA', PECAM-1,
platelet/endothelial cell adhesion molecule or naturally occurring
anti-CD38 antibody)). Thus, for example, it is possible to set a
criterion for competitiveness wherein at least about 10% relative
inhibition is detected; at least about 15% relative inhibition is
detected; or at least about 20% relative inhibition is detected
before an antibody is considered sufficiently competitive. In cases
where epitopes belonging to competing antibodies are closely
located in an antigen, competition may be marked by greater than
about 40% relative inhibition of CD38 binding (e.g., at least about
45% inhibition, such as at least about 50% inhibition, for instance
at least about 55% inhibition, such as at least about 60%
inhibition, for instance at least about 65% inhibition, such as at
least about 70% inhibition, for instance at least about 75%
inhibition, such as at least about 80% inhibition, for instance at
least about 85% inhibition, such as at least about 90% inhibition,
for instance at least about 95% inhibition, or higher level of
relative inhibition).
Competition may be considered the inverse of cross-reactivity
between a molecule and two potential binding partners. In certain
embodiments, a anti-CD38 antibody of the present invention
specifically binds to one or more residues or regions in CD38 but
also does not cross-react with other peptides, peptide regions, or
molecules, e.g., the present invention provides an anti-CD38
antibody that does not cross-react with proteins with homology to
CD38, such as BST-1 (bone marrow stromal cell antigen-1) and Mo5,
also called CD157; or anti-CD38 antibodies that do not cross-react
with CD38 in the context of normal tissue, such as tissues not
involved in multiple myeloma. Typically, a lack of cross-reactivity
means less than about 5% relative competitive inhibition between
the molecules when assessed by ELISA and/or FACS analysis using
sufficient amounts of the molecules under suitable assay
conditions.
In one embodiment, the present invention provides an anti-CD38
antibody that competes with an antibody having a V.sub.L sequence
of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2, such as
the antibody 028, for binding to CD38 or a portion thereof.
In one embodiment, the present invention provides an anti-CD38
antibody that competes with an antibody having a V.sub.L sequence
of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID NO: 7, such as
the antibody 025, for binding to CD38 or a portion thereof.
In one embodiment, the present invention provides an anti-CD38
antibody that competes with an antibody having a V.sub.L sequence
of SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12, such as
the antibody 026, for binding to CD38 or a portion thereof.
In one embodiment, the present invention provides an anti-CD38
antibody that competes with an antibody having a V.sub.L sequence
of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17, such as
the antibody 049, for binding to CD38 or a portion thereof.
In one embodiment, the present invention provides an anti-CD38
antibody that competes with an antibody having a V.sub.L sequence
of SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID NO: 22, such as
the antibody 056, for binding to CD38 or a portion thereof.
As discussed elsewhere herein, unless otherwise stated or clearly
contradicted by context, references to binding of an anti-CD38
antibody to CD38 are intended to refer to binding in any suitable
context, such as in a conformational context where the structure of
CD38 is present; or in a linear epitope context. Of course, binding
in a limited subset of such context(s) may be an important
characteristic with respect to any anti-CD38 antibody provided by
the present invention.
Additional methods for determining anti-CD38 antibody specificity
by competitive inhibition may be found in for instance Harlow et
al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds.,
Current Protocols in Immunology, Greene Publishing Assoc. and Wiley
InterScience N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92,
589-601 (1983)).
Human CD38 comprises a number of different epitopes, which may
include (1) peptide antigenic determinants that are comprised
within single peptide chains within human CD38; (2) conformational
antigenic determinants which consist of one or more noncontiguous
amino acids on a particular chain and/or amino acids present on
spatially contiguous but separate peptide chains (typically where
the respective amino acid sequences of the chains are located
disjointedly along the human CD38 polypeptide sequence); (3)
post-translational antigenic determinants which consist, either in
whole or part, of molecular structures covalently attached to human
CD38, such as carbohydrate groups; or (4) combinations of
(1)-(3).
An epitope in the context of the present invention includes any
peptide or peptide-derivative determinant capable of specific
binding to an immunoglobulin. An epitope may comprise any suitable
number of amino acids, in any suitable position (with respect to
the linear sequence of CD38), orientation (with respect to folded
CD38, or a fragment thereof), amino acid composition (and
consequently, at least in part, charge). Thus, for example, an
epitope may be composed of about 3-10 amino acids, typically 3-8
amino acids, in one or more contiguous or noncontiguous locations
with respect to the primary sequence of CD38 (for instance an
epitope may consist essentially of 2, 3, 4, 5, 6, 7, or 8 amino
acid residues distributed in 1, 2, 3, 4, or 5 noncontiguous
locations in CD38). Alternatively, for example, an epitope may be
considered to be defined by a region of about 5-40 contiguous amino
acid residues (e.g., about 7-30 amino acid residues, about 5-20
amino acid residues, or about 3-15 amino acid residues) in CD38
(solely or in combination with a portion of an adjacent CD38
domain). In some epitopes it may be the case that just one amino
acid residue or only a few amino acid residues are critical to CDR
or CDR(s) recognition (and thereby most important to anti-CD38
antibody:CD38 antigen affinity and avidity). As such, an epitope
may be characterized on the basis of one or more of such critical
residues, with the recognition that other residues may also make
some lesser contribution to the epitope. In the case of an epitope
defined by a region of amino acids, it may be that one or more
amino acids in the region make only a minor contribution or even
negligible contribution to antibody binding, such that the residue
may be subject to substitution with an appropriate different
residue without resulting in "a loss" of the epitope to at least
some anti-CD38 antibodies specific for it.
In one embodiment, the present invention provides a anti-CD38
antibody, such as an anti-CD38 antibody, that specifically binds to
a CD38 epitope that also is specifically bound by an antibody
having a V.sub.L sequence of SEQ ID NO: 27 and a V.sub.H sequence
of SEQ ID NO: 2 (such as antibody 028), or an antibody having a
V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID
NO: 7 (such as antibody 025), or an antibody having a V.sub.L
sequence of SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12
(such as antibody 026), or an antibody having a V.sub.L sequence of
SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17(such as
antibody 049), or an antibody having a V.sub.L sequence of SEQ ID
NO: 47 and a V.sub.H sequence of SEQ ID NO: 22 (such as antibody
056).
It is possible that anti-CD38 antibodies having one or more CDRs
that differ from the CDRs of an antibody having a V.sub.L sequence
of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2, or the
CDRs of an antibody having a V.sub.L sequence of SEQ ID NO: 32 and
a V.sub.H sequence of SEQ ID NO: 7, or the CDRs of an antibody
having a V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H sequence
of SEQ ID NO: 12, or the CDRs of an antibody having a V.sub.L
sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17,
or the CDRs of an antibody having a V.sub.L sequence of SEQ ID NO:
47 and a V.sub.H sequence of SEQ ID NO: 22, may still be specific
for the same epitope as an antibody having the CDRs of an antibody
having a V.sub.L sequence of SEQ ID NO: 27 and a V.sub.H sequence
of SEQ ID NO: 2, or the CDRs of an antibody having a V.sub.L
sequence of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID NO: 7,
or the CDRs of an antibody having a V.sub.L sequence of SEQ ID NO:
37 and a V.sub.H sequence of SEQ ID NO: 12, or the CDRs of an
antibody having a V.sub.L sequence of SEQ ID NO: 42 and a V.sub.H
sequence of SEQ ID NO: 17, or the CDRs of an antibody having a
V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID
NO: 22, respectively. In such cases, the anti-CD38 antibody in
question may recognize or be more specific/selective for particular
structures or regions of the epitope than the antibody having the
CDRs of an antibody having a V.sub.L sequence of SEQ ID NO: 27 and
a V.sub.H sequence of SEQ ID NO: 2, or the CDRs of an antibody
having a V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H sequence
of SEQ ID NO: 7, or the CDRs of an antibody having a V.sub.L
sequence of SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12,
or the CDRs of an antibody having a V.sub.L sequence of SEQ ID NO:
42 and a V.sub.H sequence of SEQ ID NO: 17, or the CDRs of an
antibody having a V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H
sequence of SEQ ID NO: 22 respectively.
A CD38 epitope bound by an antibody having a V.sub.L sequence of
SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2 (such as
antibody 028), or an antibody having a V.sub.L sequence of SEQ ID
NO: 32 and a V.sub.H sequence of SEQ ID NO: 7 (such as antibody
025), or an antibody having a V.sub.L sequence of SEQ ID NO: 37 and
a V.sub.H sequence of SEQ ID NO: 12 (such as antibody 026), or an
antibody having a V.sub.L sequence of SEQ ID NO: 42 and a V.sub.H
sequence of SEQ ID NO: 17(such as antibody 049), or an antibody
having a V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H sequence
of SEQ ID NO: 22 (such as antibody 056), may be identified via
standard mapping and characterization techniques, further
refinement of which may be identified by any suitable technique,
numerous examples of which are available to the skilled
artisan.
These techniques may also be used to identify and/or characterize
epitopes for anti-CD38 antibodies generally. As one example of such
mapping/characterization methods, an epitope for an anti-CD38
antibody may be determined by epitope "foot-printing" using
chemical modification of the exposed amines/carboxyls in the CD38
protein. One specific example of such a foot-printing technique is
the use of HXMS (hydrogen-deuterium exchange detected by mass
spectrometry) wherein a hydrogen/deuterium exchange of receptor and
ligand protein amide protons, binding, and back exchange occurs,
wherein the backbone amide groups participating in protein binding
are protected from back exchange and therefore will remain
deuterated. Relevant regions may be identified at this point by
peptic proteolysis, fast microbore high-performance liquid
chromatography separation, and/or electrospray ionization mass
spectrometry. See, e.g., Ehring H, Analytical Biochemistry, 267(2)
252-259 (1999) and/or Engen, J. R. and Smith, D. L. (2001) Anal.
Chem. 73, 256A-265A. Another example of a suitable epitope
identification technique is nuclear magnetic resonance epitope
mapping (NMR), where typically the position of the signals in
two-dimensional NMR spectres of the free antigen and the antigen
complexed with the antigen binding peptide, such as an antibody,
are compared. The antigen typically is selectively isotopically
labeled with .sup.15N so that only signals corresponding to the
antigen and no signals from the antigen binding peptide are seen in
the NMR-spectrum. Antigen signals originating from amino acids
involved in the interaction with the antigen binding peptide
typically will shift position in the spectres of the complex
compared to the spectres of the free antigen, and the amino acids
involved in the binding may be identified that way. See for
instance Ernst Schering Res Found Workshop. (44), 149-67 (2004),
Huang et al., Journal of Molecular Biology 281(1), 61-67 (1998) and
Saito and Patterson, Methods. 9(3), 516-24 (1996).
Epitope mapping/characterization may also be performed using mass
spectrometry methods. See for instance Downward, J Mass Spectrom.
35(4), 493-503 (2000) and Kiselar and Downard, Anal Chem. 71(9),
1792-801 (1999).
Protease digestion techniques may also be useful in the context of
epitope mapping and identification. Antigenic determinant-relevant
regions/sequences may be determined by protease digestion, e.g. by
using trypsin in a ratio of about 1:50 to CD38 overnight (O/N)
digestion at 37.degree. C. and pH 7-8, followed by mass
spectrometry (MS) analysis for peptide identification. The peptides
protected from trypsin cleavage by the CD38BP may subsequently be
identified by comparison of samples subjected to trypsin digestion
and samples incubated with CD38BP and then subjected to digestion
by e.g. trypsin (thereby revealing a foot-print for the binder).
Other enzymes like chymotrypsin, pepsin, etc. may also or
alternatively be used in a similar epitope characterization
method.
An anti-CD38 antibody which gives the significantly same result as
an antibody having a V.sub.L sequence of SEQ ID NO: 27 and a
V.sub.H sequence of SEQ ID NO: 2 (such as antibody 028), or an
antibody having a V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H
sequence of SEQ ID NO: 7 (such as antibody 025), or an antibody
having a V.sub.L sequence of SEQ ID NO: 37 and a V.sub.H sequence
of SEQ ID NO: 12 (such as antibody 026), or an antibody having a
V.sub.L sequence of SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID
NO: 17 (such as antibody 049), or an antibody having a V.sub.L
sequence of SEQ ID NO: 47 and a V.sub.H sequence of SEQ ID NO: 22
(such as antibody 056), in these measurements are deemed to be an
antibody that bind the same epitope as an antibody having a V.sub.L
sequence of SEQ ID NO: 27 and a V.sub.H sequence of SEQ ID NO: 2
(such as antibody 028), or an antibody having a V.sub.L sequence of
SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID NO: 7 (such as
antibody 025), or an antibody having a V.sub.L sequence of SEQ ID
NO: 37 and a V.sub.H sequence of SEQ ID NO: 12 (such as antibody
026), or an antibody having a V.sub.L sequence of SEQ ID NO: 42 and
a V.sub.H sequence of SEQ ID NO: 17 (such as antibody 049), or an
antibody having a V.sub.L sequence of SEQ ID NO: 47 and a V.sub.H
sequence of SEQ ID NO: 22 (such as antibody 056), respectively. See
for instance Manca, Ann Ist Super Sanita. 27(1), 15-9 (1991) for a
discussion of similar techniques.
Epitope mapping by competitive binding to CD38 with two antibodies
where one is biotinylated is another method for identifying
relevant antigenic determinant regions. The binding of antibodies
to linear and looped peptides of CD38 by a PEPSCAN-based
enzyme-linked immuno assay is another method for identifying
relevant antigenic determinant regions, see for instance
Slootstra-JW et al. Mol-Divers. 1, 87-96 (1996).
Site directed mutagenesis is another method for identifying
relevant antigenic determinant regions, see for instance Polyak and
Deans, Blood 99, 3956-3962 (2002). Various phage display techniques
may also be used to identify epitopes. See for instance Wang and
Yu, Curr Drug Targets. 5(1), 1-15 (2004), Burton, Immunotechnology.
1(2), 87-94 (1995 Aug), Cortese et al., Immunotechnology. 1(2),
87-94 (1995) and Irving et al., Curr Opin Chem Biol. 5(3), 314-24
(2001). Consensus epitopes may also be identified through modified
phage display-related techniques (see,
http://www.cs.montana.edu/.about.mumey/papers/jcb03.pdf) for
discussion.
Other methods potentially helpful in mapping epitopes include
crystallography techniques, X-ray diffraction techniques (such as
the X-ray diffraction/sequence study techniques developed by Poljak
and others in the 1970s-1980s), and the application of Multipin
Peptide Synthesis Technology. Computer-based methods such as
sequence analysis and three dimensional structure analysis and
docking may also be used to identify antigenic determinants. For
example, an epitope may also be determined by molecular modeling
using a structure of CD38 with docking of the structure of the Fab
fragment of the individual monoclonal antibody. These and other
mapping methods are discussed in Epitope Mapping A Practical
Approach (Westwood and Hay Eds.) 2001 Oxford University Press.
In one embodiment, the present invention provides an anti-CD38
antibody having substantially the same specific CD38-binding
characteristics of one or more mAbs selected from an antibody
having a V.sub.L sequence of SEQ ID NO: 27 and a V.sub.H sequence
of SEQ ID NO: 2 (such as antibody 028), or an antibody having a
V.sub.L sequence of SEQ ID NO: 32 and a V.sub.H sequence of SEQ ID
NO: 7 (such as antibody 025), or an antibody having a V.sub.L
sequence of SEQ ID NO: 37 and a V.sub.H sequence of SEQ ID NO: 12
(such as antibody 026), or an antibody having a V.sub.L sequence of
SEQ ID NO: 42 and a V.sub.H sequence of SEQ ID NO: 17 (such as
antibody 049), or an antibody having a V.sub.L sequence of SEQ ID
NO: 47 and a V.sub.H sequence of SEQ ID NO: 22 (such as antibody
056).
Mapping studies have indicated that several monoclonal antibodies
raised against human CD38 bind to epitopes in the C-terminal region
of CD38 (220-296) (Hoshino et al. and Ferrero et al.). Within this
region three amino acid differences have been found between the
human and the cynomolgus CD38 sequence: T237, Q272 and S274 in
humans correspond to A238, R273 and F275 in cynomolgus. A limited
number of amino acid differences exist between the human and the
monkey CD38 sequence, for instance in the carboxyterminal part to
the protein, for instance the following three amino acid
differences between the human and the cynomolgus CD38 sequence:
T237, Q272 and S274 in human CD38s correspond to A238, R273 and
F275 in cynomolgus monkey CD38 (compare SEQ ID No.21 and SEQ ID
No.22).
The antibodies of the present invention do not bind to human CD38
mutants wherein aspartic acid in position 202 has been substituted
with a glycine to the same degree that it binds to human CD38. The
present invention provides antibodies, which bind to human CD38 and
which binds to a mutant human CD38, wherein the serine residue in
position 274 has been substituted with a phenylalanine residue. The
antibodies of the present invention also bind to human CD38 mutants
wherein glutamine in position 272 has been substituted with an
arginine. The antibodies of the present invention also bind to
human CD38 mutants wherein the threonine in position 237 has been
substituted with an alanine.
The term "do not bind to the same degree" should be interpreted so
that the binding of the antibody to the mutant human CD38 is
significantly lower than the binding of the antibody to the wild
type human CD38. The term "bind to the same degree" should be
interpreted so that the binding of the antibody to the mutant human
CD38 is substantially of the same order as the binding of the
antibody to the wild type human CD38. The binding of a peptide to
the CD38 molecules (wild type and mutant) may be determined in a
number of ways and it is within the common general knowledge of a
person skilled in the art to determine whether the binding to the
mutant is "significantly lower" than the binding to the wild type.
A large number of different techniques for determining the binding
of a peptide to another peptide are available to the person skilled
in the art, for example ELISA, radioimmunoassay, BIAcore or flow
cytometry.
One method of determining the binding is by determining the
EC.sub.50 of the binding of the antibody to the mutant protein and
to the wild type protein and then comparing the values obtained.
Another method of determining the binding is by examining the
magnitude of binding at saturating concentration (for instance the
plateau of binding signal), or by determining kinetic rate
constants k.sub.on and k.sub.off for example by BIAcore.
In one embodiment, the binding of the antibody in question to the
CD38 proteins (mutant or wild type) is by use of an ELISA as
described in Example 4.
In a further embodiment, the antibody of the invention comprises a
human heavy chain variable region (VH) CDR3 sequence comprising: an
amino acid sequence selected from the group consisting of: SEQ ID
NOs: 5, 10, 15, 20 and 25, or a variant of any of said sequences,
wherein said variant preferably only has conservative amino acid
modifications.
In one embodiment, said variant consists essentially of a sequence
having at least about 50%, such as at least 60%, for instance at
least about 70%, such as at least about 75%, for instance at least
about 80%, such as at least about 85%, for instance at least about
90%, such as at least about 95% amino acid sequence identity to a
sequence according to any one of SEQ ID Nos: 5, 10, 15, 20 and
25.
In a further embodiment, said variant has at most 1, 2 or 3
amino-acid modifications, e.g. amino acid substitutions, preferably
conservative substitutions as compared to said sequence.
In a preferred embodiment, said antibody comprises a human heavy
chain variable region CDR3 sequence comprising an amino acid
sequence selected from the group consisting of: SEQ ID NOs: 5, 10,
15, 20 and 25.
In an even further embodiment, the antibody of the invention
comprises: a VH region comprising the CDR1, 2 and 3 sequences of
SEQ ID NO: 3, 4 and 5; or a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 8, 9 and 10; or a VH region comprising the
CDR1, 2 and 3 sequences of SEQ ID NO: 13, 14 and 15; or a VH region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 18, 19 and 20;
or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO:
23, 24 and 25; or a variant of any said VH regions, wherein said
variant preferably only has conservative amino-acid
substitutions.
In one embodiment, said variant comprises a VH CDR1 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:3, 8, 13, 18 or 23.
In one embodiment, said variant comprises a VH CDR2 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:4, 9, 14, 19 or 24.
In one embodiment, said variant comprises a VH CDR3 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos: 5, 10, 15, 20 or 25.
In another embodiment, the antibody the antibody comprises a) a VL
CDR3 region having the sequence as set forth in SEQ ID NO: 30 and a
VH CDR3 region having a sequence selected from the group consisting
of SEQ ID NO: 5, b) a VL CDR3 region having the sequence as set
forth in SEQ ID NO: 35 and a VH CDR3 region having the sequence as
set forth in SEQ ID NO: 10, c) a VL CDR3 region having the sequence
as set forth in SEQ ID NO: 40 and a VH CDR3 region having the
sequence as set forth in SEQ ID NO: 15, d) a VL CDR3 region having
the sequence as set forth in SEQ ID NO: 45 and a VH CDR3 region
having the sequence as set forth in SEQ ID NO: 20, e) a VL CDR3
region having the sequence as set forth in SEQ ID NO: 50 and a VH
CDR3 region having the sequence as set forth in SEQ ID NO: 25, f) a
variant of any of the above, wherein said variant preferably only
has conservative substitutions in said sequences
In one embodiment, said variant comprises a VH CDR3 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos: 5, 10, 15, 20 or 25. In one embodiment, said variant comprises
a VL CDR3 which consists essentially of a sequence having at least
about 50%, such as at least 60%, for instance at least about 70%,
such as at least about 75%, for instance at least about 80%, such
as at least about 85%, for instance at least about 90%, such as at
least about 95% amino acid sequence identity to a sequence
according to any one of SEQ ID Nos: 30, 35, 40, 45 or 50;
In a further embodiment, the antibody of the invention comprises: a
VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 3, 4
and 5, and a VL region comprising the CDR 3 sequence of SEQ ID NO:
30; or a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO: 8, 9 and 10, and a VL region comprising the CDR3 sequence of
SEQ ID NO: 35; or a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 13, 14 and 15, and and a VL region
comprising the CDR3 sequence of SEQ ID NO: 40; or a VH region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 18, 19 and 20,
and a VL region comprising the CDR3 sequence of SEQ ID NO: 45; or a
VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 23,
24 and 25, and a VL region comprising the CDR3 sequence of SEQ ID
NO: 50; or a variant of any of said antibodies, wherein said
variant preferably only has conservative amino-acid substitutions
in said sequences.
In one embodiment, said variant comprises a VH CDR1 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos: 3, 8, 13, 18 or 23.
In one embodiment, said variant comprises a VH CDR2 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos: 4, 9, 14, 19 or 24.
In one embodiment, said variant comprises a VH CDR3 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:5, 10, 15, 20 or 25.
In one embodiment, said variant comprises a VL CDR1 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:28, 33, 38, 43 or 48.
In one embodiment, said variant comprises a VL CDR2 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:29, 34, 39, 44 or 49.
In one embodiment, said variant comprises a VL CDR3 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:30, 35, 40, 45, or 50.
In a further embodiment, the antibody of the invention comprises: a
VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 3, 4
and 5 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ
ID NO: 28, 29 and 30; or a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 8, 9 and 10 and a VL region comprising the
CDR1, 2 and 3 sequences of SEQ ID NO: 33, 34 and 35; or a VH region
comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 13, 14 and 15
and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID
NO: 38, 39 and 40; or a VH region comprising the CDR1, 2 and 3
sequences of SEQ ID NO: 18, 19 and 20 and a VL region comprising
the CDR1, 2 and 3 sequences of SEQ ID NO: 43, 44 and 45; or a VH
region comprising the CDR1, 2 and 3 sequences of SEQ ID NO: 23, 24
and 25 and a VL region comprising the CDR1, 2 and 3 sequences of
SEQ ID NO: 48, 49 and 50; or a variant of any of said antibodies,
wherein said variant preferably only has conservative amino acid
modifications in said sequences.
In one embodiment, said variant comprises a VH CDR1 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos: 3, 8, 13, 18 or 23;
In one embodiment, said variant comprises a VH CDR2 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:4, 9, 14, 19 or 24.
In one embodiment, said variant comprises a VH CDR3 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:5, 10, 15, 20 or 25.
In one embodiment, said variant comprises a VL CDR1 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:28, 33, 38, 43 or 48.
In one embodiment, said variant comprises a VL CDR2 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:29, 34, 39, 44 or 49.
In one embodiment, said variant comprises a VL CDR3 which consists
essentially of a sequence having at least about 50%, such as at
least 60%, for instance at least about 70%, such as at least about
75%, for instance at least about 80%, such as at least about 85%,
for instance at least about 90%, such as at least about 95% amino
acid sequence identity to a sequence according to any one of SEQ ID
Nos:30, 35, 40, 45 or 50.
In an even further embodiment, the antibody of the invention
comprises: a VH region comprising the sequence of SEQ ID NO: 2 and
a VL region comprising the sequence of SEQ ID NO: 27; or a VH
region comprising the sequence of SEQ ID NO: 7 and a VL region
comprising the sequence of SEQ ID NO: 32; or a VH region comprising
the sequence of SEQ ID NO: 12 and a VL region comprising the
sequence of SEQ ID NO: 37; or a VH region comprising the sequence
of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID
NO: 42; or a VH region comprising the sequence of SEQ ID NO: 22 and
a VL region comprising the sequence of SEQ ID NO: 47; or a variant
of any of the above, wherein said variant preferably only has
conservative modifications.
In one embodiment, said variant comprises a VH region which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID Nos:2, 7, 12, 17 or 22.
In one embodiment, said variant comprises a VL region which
consists essentially of a sequence having at least about 50%, such
as at least 60%, for instance at least about 70%, such as at least
about 75%, for instance at least about 80%, such as at least about
85%, for instance at least about 90%, such as at least about 95%
amino acid sequence identity to a sequence according to any one of
SEQ ID NO: 27, 32, 37, 42 or 47.
In a further embodiment the antibody of the present invention
comprises a VH having at least 80% identity, such as 90%, or 95%,
or 97%, or 98%, or 99% identity to a VH region sequence selected
from the group consisting of: SEQ ID NO: 2, 7, 12, 17 or 22.
In a further embodiment the antibody of the present invention
comprises a VL having at least 80% identity, such as 90%, or 95%,
or 97%, or 98%, or 99% identity to a VL region sequence selected
from the group consisting of: SEQ ID NO: 27, 32, 37, 42 or 47.
In an even further embodiment, the antibody of the invention
comprises a VH region selected from the group consisting of: SEQ ID
NO: 2, 7, 12, 17 or 22.
In an even further embodiment, the antibody of the invention
comprises a VL region selected from the group consisting of: SEQ ID
NO: 27, 32, 37, 42 or 47.
In an even further embodiment, the antibody of the invention
comprises: a VH region comprising the sequence of SEQ ID NO: 2 and
a VL region comprising the sequence of SEQ ID NO: 27; or a VH
region comprising the sequence of SEQ ID NO: 7 and a VL region
comprising the sequence of SEQ ID NO: 32; or a VH region comprising
the sequence of SEQ ID NO: 12 and a VL region comprising the
sequence of SEQ ID NO: 37; or a VH region comprising the sequence
of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID
NO: 42; or a VH region comprising the sequence of SEQ ID NO: 22 and
a VL region comprising the sequence of SEQ ID NO: 47; or
The present invention also, in one aspect, provides anti-CD38
antibodies which are characterized with respect to their ability to
compete with an antibody having: a VH region comprising the
sequence of SEQ ID NO: 2 and a VL region comprising the sequence of
SEQ ID NO: 27; or a VH region comprising the sequence of SEQ ID NO:
7 and a VL region comprising the sequence of SEQ ID NO: 32; or a VH
region comprising the sequence of SEQ ID NO: 12 and a VL region
comprising the sequence of SEQ ID NO: 37; or a VH region comprising
the sequence of SEQ ID NO: 17 and a VL region comprising the
sequence of SEQ ID NO: 42; or a VH region comprising the sequence
of SEQ ID NO: 22 and a VL region comprising the sequence of SEQ ID
NO: 47.
The present invention also relates to provides anti-CD38 antibodies
which bind to the same epitope as an antibody having: a VH region
comprising the sequence of SEQ ID NO: 2 and a VL region comprising
the sequence of SEQ ID NO: 27; or a VH region comprising the
sequence of SEQ ID NO: 7 and a VL region comprising the sequence of
SEQ ID NO: 32; or a VH region comprising the sequence of SEQ ID NO:
12 and a VL region comprising the sequence of SEQ ID NO: 37; or a
VH region comprising the sequence of SEQ ID NO: 17 and a VL region
comprising the sequence of SEQ ID NO: 42; or a VH region comprising
the sequence of SEQ ID NO: 22 and a VL region comprising the
sequence of SEQ ID NO: 47.
The antibody of the invention may be of any isotype. The choice of
isotype typically will be guided by the desired effector functions,
such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3,
and IgG4. Either of the human light chain constant regions, kappa
or lambda, may be used. If desired, the class of an anti-CD38
antibody of the present invention may be switched by known methods.
For example, an antibody of the present invention that was
originally IgM may be class switched to an IgG antibody of the
present invention. Further, class switching techniques may be used
to convert one IgG subclass to another, for instance from IgG1 to
IgG2. Thus, the effector function of the antibodies of the present
invention may be changed by isotype switching to, e.g., an IgG1,
IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various
therapeutic uses. In one embodiment an antibody of the present
invention is an IgG1 antibody, for instance an IgG1,.kappa..
In one embodiment, the antibody of the invention is a full-length
antibody. In another embodiment, the antibody of the invention is
an antibody fragment or a single-chain antibody.
Antibody fragments may e.g. be obtained by fragmentation using
conventional techniques, and the fragments screened for utility in
the same manner as described herein for whole antibodies. For
example, F(ab').sub.2 fragments may be generated by treating
antibody with pepsin. The resulting F(ab').sub.2 fragment may be
treated to reduce disulfide bridges to produce Fab' fragments. Fab
fragments may be obtained by treating an IgG antibody with papain;
Fab' fragments may be obtained with pepsin digestion of IgG
antibody. A F(ab') fragment may also be produced by binding Fab'
described below via a thioether bond or a disulfide bond. A Fab'
fragment is an antibody fragment obtained by cutting a disulfide
bond of the hinge region of the F(ab').sub.2. A Fab' fragment may
be obtained by treating a F(ab').sub.2 fragment with a reducing
agent, such as dithiothreitol. Antibody fragments may also be
generated by expression of nucleic acids encoding such fragments in
recombinant cells (see for instance Evans et al., J. Immunol. Meth.
184, 123-38 (1995)). For example, a chimeric gene encoding a
portion of a F(ab').sub.2 fragment could include DNA sequences
encoding the C.sub.H1 domain and hinge region of the H chain,
followed by a translational stop codon to yield such a truncated
antibody fragment molecule.
In one embodiment, the anti-CD38 antibody is a monovalent antibody,
preferably a monovalent antibody as described in WO2007059782
(Genmab) (incorporated herein by reference). Accordingly, in one
embodiment, the antibody is a monovalent antibody, wherein said
anti-CD38 antibody is constructed by a method comprising: i)
providing a nucleic acid construct encoding the light chain of said
monovalent antibody, said construct comprising a nucleotide
sequence encoding the VL region of a selected antigen specific
anti-CD38 antibody and a nucleotide sequence encoding the constant
CL region of an Ig, wherein said nucleotide sequence encoding the
VL region of a selected antigen specific antibody and said
nucleotide sequence encoding the CL region of an Ig are operably
linked together, and wherein, in case of an IgG1 subtype, the
nucleotide sequence encoding the CL region has been modified such
that the CL region does not contain any amino acids capable of
forming disulfide bonds or covalent bonds with other peptides
comprising an identical amino acid sequence of the CL region in the
presence of polyclonal human IgG or when administered to an animal
or human being; ii) providing a nucleic acid construct encoding the
heavy chain of said monovalent antibody, said construct comprising
a nucleotide sequence encoding the VH region of a selected antigen
specific antibody and a nucleotide sequence encoding a constant CH
region of a human Ig, wherein the nucleotide sequence encoding the
CH region has been modified such that the region corresponding to
the hinge region and, as required by the Ig subtype, other regions
of the CH region, such as the CH3 region, does not comprise any
amino acid residues which participate in the formation of
disulphide bonds or covalent or stable non-covalent inter-heavy
chain bonds with other peptides comprising an identical amino acid
sequence of the CH region of the human Ig in the presence of
polyclonal human IgG or when administered to an animal human being,
wherein said nucleotide sequence encoding the VH region of a
selected antigen specific antibody and said nucleotide sequence
encoding the CH region of said Ig are operably linked together;
iii) providing a cell expression system for producing said
monovalent antibody; iv) producing said monovalent antibody by
co-expressing the nucleic acid constructs of (i) and (ii) in cells
of the cell expression system of (iii).
Similarly, in one embodiment, the anti-CD38 antibody is a
monovalent antibody, which comprises (i) a variable region of an
antibody of the invention as described herein or an antigen binding
part of the said region, and (ii) a C.sub.H region of an
immunoglobulin or a fragment thereof comprising the C.sub.H2 and
C.sub.H3 regions, wherein the C.sub.H region or fragment thereof
has been modified such that the region corresponding to the hinge
region and, if the immunoglobulin is not an IgG4 subtype, other
regions of the C.sub.H region, such as the C.sub.H3 region, do not
comprise any amino acid residues, which are capable of forming
disulfide bonds with an identical C.sub.H region or other covalent
or stable non-covalent inter-heavy chain bonds with an identical
C.sub.H region in the presence of polyclonal human IgG.
In a further embodiment, the heavy chain of the monovalent
anti-CD38 antibody has been modified such that the entire hinge has
been deleted.
In a further embodiment, said monovalent antibody is of the IgG4
subtype, but the C.sub.H3 region has been modified so that one or
more of the following amino acid substitutions have been made: Thr
(T) in position 366 has been replaced by Ala (A); Leu (L) in
position 368 has been replaced by Ala (A); Leu (L) in position 368
has been replaced by Val (V); Phe (F) in position 405 has been
replaced by Ala (A); Phe (F) in position 405 has been replaced by
Leu (L); Tyr (Y) in position 407 has been replaced by Ala (A); Arg
(R) in position 409 has been replaced by Ala (A).
In another further embodiment, the sequence of said monovalent
antibody has been modified so that it does not comprise any
acceptor sites for N-linked glycosylation.
Anti-CD38 antibodies of the invention also include single chain
antibodies. Single chain antibodies are peptides in which the heavy
and light chain Fv regions are connected. In one embodiment, the
present invention provides a single-chain Fv (scFv) wherein the
heavy and light chains in the Fv of an anti-CD38 antibody of the
present invention are joined with a flexible peptide linker
(typically of about 10, 12, 15 or more amino acid residues) in a
single peptide chain. Methods of producing such antibodies are
described in for instance U.S. Pat. No. 4,946,778, Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et
al., Science 242, 423-426 (1988), Huston et al., PNAS USA 85,
5879-5883 (1988) and McCafferty et al., Nature 348, 552-554 (1990).
The single chain antibody may be monovalent, if only a single
V.sub.H and V.sub.L are used, bivalent, if two V.sub.H and V.sub.L
are used, or polyvalent, if more than two V.sub.H and V.sub.L are
used.
In one embodiment, the anti-CD38 antibody of the invention is an
effector-function-deficient antibody. Such antibodies are
particularly useful when the antibody is for use in stimulation and
demping of the immune system through blocking of the inhibitory
effects of CD38. For such applications, it may be advantageous that
the antibody has no effector functions, such as ADCC, as this may
lead to undesired cytotoxicity.
In one embodiment, the effector-function-deficient anti-CD38
antibody is a stabilized IgG4 antibody. Examples of suitable
stabilized IgG4 antibodies are antibodies, wherein arginine at
position 409 in a heavy chain constant region of human IgG4, which
is indicated in the EU index as in Kabat et al., is substituted
with lysine, threonine, methionine, or leucine, preferably lysine
(described in WO2006033386 (Kirin)). Preferably, said antibody
comprises a Lys or Ala residue at the position corresponding to 409
or the CH3 region of the antibody has been replaced by the CH3
region of human IgG1, of human IgG2 or of human IgG3.
In a further embodiment. the stabilized IgG4 anti-CD38 antibody is
an IgG4 antibody comprising a heavy chain and a light chain,
wherein said heavy chain comprises a human IgG4 constant region
having a residue selected from the group consisting of: Lys, Ala,
Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions, but does not comprise a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the CH3 region of the antibody has been
replaced by the CH3 region of human IgG1, of human IgG2 or of human
IgG3.
In an even further embodiment. the stabilized IgG4 anti-CD38
antibody is an IgG4 antibody comprising a heavy chain and a light
chain, wherein said heavy chain comprises a human IgG4 constant
region having a residue selected from the group consisting of: Lys,
Ala, Thr, Met and Leu at the position corresponding to 409 and/or a
residue selected from the group consisting of: Ala, Val, Gly, Ile
and Leu at the position corresponding to 405, and wherein said
antibody optionally comprises one or more further substitutions,
deletions and/or insertions and wherein said antibody comprises a
Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, said
antibody comprises a Lys or Ala residue at the position
corresponding to 409 or the CH3 region of the antibody has been
replaced by the CH3 region of human IgG1, of human IgG2 or of human
IgG3.
In a further embodiment, the effector-function-deficient anti-CD38
antibody is an antibody of a non-IgG4 type, e.g. IgG1, IgG2 or IgG3
which has been mutated such that the ability to mediate effector
functions, such as ADCC, has been reduced or even eliminated.
Examples of such mutations have e.g. been described in Dall'Acqua
WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J
Virol.;75(24):12161-12168 (2001).
Conjugates
In a further embodiment. the antibody of the invention is
conjugated to another moiety, such as a cytotoxic moiety, a
radioisotope or a drug.
Such antibodies may be produced by chemically conjugating the other
moiety to the N-terminal side or C-terminal side of the anti-CD38
antibody or fragment thereof (e.g., an anti-CD38 antibody H chain,
L chain, or anti-CD38 specific/selective fragment thereof) (see,
e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu,
published by Chijin Shokan (1994)). Such conjugated antibody
derivatives may also be generated by conjugation at internal
residues or sugars, where appropriate.
Anti-CD38 antibodies described herein may also be modified by
inclusion of any suitable number of modified amino acids.
Suitability in this context is generally determined by the ability
to at least substantially retain CD38 selectivity and/or
specificity associated with the non-derivatized parent anti-CD38
antibody. The inclusion of one or more modified amino acids may be
advantageous in, for example, increasing polypeptide serum
half-life, reducing polypeptide antigenicity, or increasing
polypeptide storage stability. Amino acid(s) are modified, for
example, co-translationally or post-translationally during
recombinant production (e. g., N-linked glycosylation at N--X--S/T
motifs during expression in mammalian cells) or modified by
synthetic means. Non-limiting examples of a modified amino acid
include a glycosylated amino acid, a sulfated amino acid, a
prenylated (e. g., farnesylated, geranylgeranylated) amino acid, an
acetylated amino acid, an acylated amino acid, a PEGylated amino
acid, a biotinylated amino acid, a carboxylated amino acid, a
phosphorylated amino acid, and the like. References adequate to
guide one of skill in the modification of amino acids are replete
throughout the literature. Example protocols are found in Walker
(1998) Protein Protocols On Cd-Rom, Humana Press, Towata, N.J.
Anti-CD38 antibodies may also be chemically modified by covalent
conjugation to a polymer to for instance increase their circulating
half-life. Exemplary polymers, and methods to attach them to
peptides, are illustrated in for instance U.S. Pat. Nos. 4,766,106,
4,179,337, 4,495,285 and 4,609,546.
In one embodiment, the present invention provides an anti-CD38
antibody that is conjugated to a second molecule that is selected
from a radionuclide, an enzyme, an enzyme substrate, a cofactor, a
fluorescent marker, a chemiluminescent marker, a peptide tag, or a
magnetic particle. In one embodiment, an anti-CD38 antibody may be
conjugated to one or more antibody fragments, nucleic acids
(oligonucleotides), nucleases, hormones, immunomodulators,
chelators, boron compounds, photoactive agents, dyes, and the like.
These and other suitable agents may be coupled either directly or
indirectly to an anti-CD38 antibody of the present invention. One
example of indirect coupling of a second agent is coupling by a
spacer moiety.
In one embodiment, anti-CD38 antibodies comprising one or more
radiolabeled amino acids are provided. A radiolabeled anti-CD38
antibody may be used for both diagnostic and therapeutic purposes
(conjugation to radiolabeled molecules is another possible
feature). Nonlimiting examples of labels for polypeptides include,
but are not limited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 1251,
1311, and 186Re. Methods for preparing radiolabeled amino acids and
related peptide derivatives are known in the art (see for instance
Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d
edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S.
Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (US RE35,500),
U.S. Pat. Nos. 5,648,471 and 5,697,902.
In one embodiment, the anti-CD38 antibody of the invention is
conjugated to a radioisotope or to a radioisotope-containing
chelate. For example, the anti-CD38 antibody can be conjugated to a
chelator linker, e.g. DOTA, DTPA or tiuxetan, which allows for the
anti-CD38 antibody to be complexed with a radioisotope. The
anti-CD38 antibody may also or alternatively comprise or be
conjugated to one or more radiolabeled amino acids or other
radiolabeled molecule. A radiolabeled anti-CD38 antibody may be
used for both diagnostic and therapeutic purposes. Non-limiting
examples of radioisotopes include 3H, 14C, 15N, 35S, 90Y, 99Tc,
125I, 111In, 131I, 186Re, 213Bs, 225Ac and 227Th.
In one embodiment, the anti-CD38 antibody of the invention is
conjugated to auristatins or auristatin peptide analogs and
derivates (U.S. Pat. Nos. 5,635,483; 5,780,588). Auristatins have
been shown to interfere with microtubule dynamics, GTP hydrolysis
and nuclear and cellular division (Woyke et al (2001) Antimicrob.
Agents and Chemother. 45(12): 3580-3584) and have anticancer (U.S.
Pat. No. 5,663,149) and antifungal activity (Pettit et al., (1998)
Antimicrob. Agents and Chemother. 42:2961-2965. The auristatin drug
moiety may be attached to the antibody, via an linker, through the
N (amino) terminus or the C (terminus) of the peptidic drug
moiety.
Exemplary auristatin embodiments include the N-terminus-linked
monomethyl auristatin drug moieties DE and DF, disclosed in Senter
et al., Proceedings of the American Association for Cancer
Research. Volume 45, abstract number 623, presented Mar. 28, 2004
and described in US 2005/0238648).
An exemplary auristatin embodiment is MMAE (monomethyl auristatin
E), wherein the wavy line indicates the covalent attachment to the
linker (L) of an antibody drug conjugate:
##STR00001##
Another exemplary auristatin embodiment is MMAF (monomethyl
auristatin F), wherein the wavy line indicates the covalent
attachment to a linker (L) of an antibody drug conjugate
(US2005/0238649):
##STR00002##
The anti-CD38 antibody drug conjugates according to the invention
comprise a linker unit between the cytostatic drug unit and the
antibody unit. In some embodiments, the linker is cleavable under
intracellular conditions, such that the cleavage of the linker
releases the drug unit from the antibody in the intracellular
environment. In yet another embodiment, the linker unit is not
cleavable and the drug is for instance released by antibody
degradation. In some embodiments, the linker is cleavable by a
cleavable agent that is present in the intracellular environment
(e. g. within a lysosome or endosome or caveola). The linker can
be, e. g. a peptidyl linker that is cleaved by an intracellular
peptidase or protease enzyme, including but not limited to, a
lysosomal or endosomal protease. In some embodiments, the peptidyl
linker is at least two amino acids long or at least three amino
acids long. Cleaving agents can include cathepsins B and D and
plasmin, all of which are known to hydrolyze dipeptide drug
derivatives resulting in the release of active drug inside the
target cells (see e. g. Dubowchik and Walker, 1999, Pharm.
Therapeutics 83:67-123). In a specific embodiment, the peptidyl
linker cleavable by an intracellular protease is a Val-Cit
(valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine)
linker (see e.g. U.S. Pat. No. 6,214,345, which describes the
synthesis of doxorubicin with the Val-Cit linker). An advantage of
using intracellular proteolytic release of the therapeutic agent is
that the agent is typically attenuated when conjugated and the
serum stabilities of the conjugates are typically high.
In yet another embodiment, the linker unit is not cleavable and the
drug is released by antibody degradation (see US 2005/0238649).
Typically, such a linker is not substantially sensitive to the
extracellular environment. As used herein, "not substantially
sensitive to the extracellular environment" in the context of a
linker means that no more than 20%, typically no more than about
15%, more typically no more than about 10%, and even more typically
no more than about 5%, no more than about 3%, or no more than about
1% of the linkers, in a sample of antibody drug conjugate compound,
are cleaved when the antibody drug conjugate compound presents in
an extracellular environment (e.g. plasma). Whether a linker is not
substantially sensitive to the extracellular environment can be
determined for example by incubating with plasma the antibody drug
conjugate compound for a predetermined time period (e.g. 2, 4, 8,
16 or 24 hours) and then quantitating the amount of free drug
present in the plasma.
Additional exemplary embodiments comprising MMAE or MMAF and
various linker components have the following structures (wherein Ab
means antibody and p, representing the drug-loading (or average
number of cytostatic drugs per molecule), is 1 to about 8).
Examples where a cleavable linker is combined with an auristatin
include vcMMAF and vcMMAE (vc is the abbreviation for the Val-Cit
(valine-citruline) based linker):
##STR00003##
Other examples include auristatins combined with a non-cleavable
linker, such as mcMMAF. (mc is an abbreviation of maleimido
caproyl):
##STR00004##
The cytostatic drug loading is represented by p and is the average
number of cytostatic drug moieties per antibody in a molecule (also
designated as the drug to antibody ratio, DAR). The cytostatic drug
loading may range from 1 to 20 drug moieties per antibody and may
occur on amino acids with useful functional groups such as, but not
limited to, amino or sulfhydryl groups, as in lysine or
cysteine.
Depending on the way of conjugation, p may be limited by the number
of attachment sites on the antibody, for example where the
attachment is a cysteine thiol, as in the present invention.
Generally, antibodies do not contain many free and reactive
cysteine thiol groups which may be linked to a drug moiety as most
cysteine thiol residues in antibodies exist as disulfide bridges.
Therefore, in certain embodiments, an antibody may be reduced with
reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or fully reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, the drug loading for an ADC of the invention ranges
from 1 to about 8, as a maximum of 8 free cysteine thiol groups
becomes available after (partial) reduction of the antibody (there
are 8 cysteines involved in inter-chain disulfide bonding).
In one embodiment, the drug linker moiety is vcMMAE. The vcMMAE
drug linker moiety and conjugation methods are disclosed in
WO2004010957, U.S. Pat. Nos. 7,659,241, 7,829,531, 7,851,437 and
U.S. Ser. No. 11/833,028 (Seattle Genetics, Inc.), (which are
incorporated herein by reference), and the vcMMAE drug linker
moiety is bound to the anti-CD38 antibodies at the cysteines using
a method similar to those disclosed in therein.
In one embodiment, the drug linker moiety is mcMMAF. The mcMMAF
drug linker moiety and conjugation methods are disclosed in U.S.
Pat. No. 7,498,298, U.S. Ser. No. 11/833,954, and WO2005081711
(Seattle Genetics, Inc.) (which are incorporated herein by
reference), and the mcMMAF drug linker moiety is bound to the
anti-CD38 antibodies at the cysteines using a method similar to
those disclosed in therein.
Upon purifying the anti-CD38 antibody drug conjugates they may be
formulated into pharmaceutical compositions using well known
pharmaceutical carriers or excipients.
In one embodiment, an anti-CD38 antibody is conjugated to a
functional nucleic acid molecule. Functional nucleic acids include
antisense molecules, interfering nucleic acid molecules (e.g.,
siRNA molecules), aptamers, ribozymes, triplex forming molecules,
and external guide sequences. External guide sequences (EGSs) are
molecules that bind a target nucleic acid molecule forming a
complex that is recognized by RNase P, which cleaves the target
molecule. The functional nucleic acid molecules may act as
effectors, inhibitors, modulators, and stimulators of a specific
activity possessed by a target molecule, or the functional nucleic
acid molecules may possess a de novo activity independent of any
other molecules. A representative sample of methods and techniques
which aid in the design and use of antisense molecules may be found
in the following non-limiting list of US patents: U.S. Pat. Nos.
5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607,
5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088,
5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898,
6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319
and 6,057,437.
Any method known in the art for conjugating the anti-CD38 antibody
to the conjugated molecule(s), such as those described above, may
be employed, including those methods described by Hunter et al.,
Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974),
Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J.
Histochem. and Cytochem. 30, 407 (1982). Linkage/conjugation may be
accomplished in any suitable way. For example, a covalent linkage
may take the form of a disulfide bond (if necessary and suitable,
an anti-CD38 antibody could be engineered to contain an extra
cysteine codon. A toxin molecule, derivatized with a sulfhydryl
group reactive with the cysteine of the modified anti-CD38
antibody, may form an immunoconjugate with the anti-CD38 antibody.
Alternatively, a sulfhydryl group may be introduced directly to an
anti-CD38 antibody using solid phase polypeptide techniques. For
example, the introduction of sulfhydryl groups into peptides is
described by Hiskey, Peptides 3, 137 (1981). The introduction of
sulfhydryl groups into proteins is described in Maasen et al., Eur.
J. Biochem. 134, 32 (1983).
Numerous types of cytotoxic compounds may be joined to proteins
through the use of a reactive group on the cytotoxic compound or
through the use of a cross-linking agent. A common reactive group
that will form a stable covalent bond in vivo with an amine is
isothiocyanate (Means et al., Chemical modifications of proteins
(Holden-Day, San Francisco 1971) pp. 105-110). This group
preferentially reacts with the .epsilon.-amine group of lysine.
Maleimide is a commonly used reactive group to form a stable in
vivo covalent bond with the sulfhydryl group on cysteine (Ji.,
Methods Enzymol 91, 580-609 (1983)). Monoclonal antibodies
typically are incapable of forming covalent bonds with radiometal
ions, but they may be attached to the antibody indirectly through
the use of chelating agents that are covalently linked to the
antibodies. Chelating agents may be attached through amines (Meares
et al., Anal. Biochem. 142, 68-78 (1984)) and sulfhydral groups
(Koyama, Chem. Abstr. 120, 217262t (1994)) of amino acid residues
and also through carbohydrate groups (Rodwell et al., PNAS USA 83,
2632-2636 (1986), Quadri et al., Nucl. Med. Biol. 20, 559-570
(1993)). A therapeutic or diagnostic agent may also or
alternatively be attached at the hinge region of a reduced antibody
component via disulfide bond formation.
In one embodiment, the present invention provides an anti-CD38
antibody, such as a human anti-CD38 antibody, conjugated to a
therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug,
an immunosuppressant, or a radioisotope. Such conjugates are
referred to herein as "immunoconjugates". Immunoconjugates which
include one or more cytotoxins are referred to as "immunotoxins". A
cytotoxin or cytotoxic agent includes any agent that is detrimental
to (e.g., kills) cells. For a description of these classes of drugs
which are well known in the art, and their mechanisms of action,
see Goodman et al., Goodman and Gilman's The Pharmacological Basis
Of Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
Additional techniques relevant to the preparation of antibody
immunotoxins are provided in for instance Vitetta, Immunol. Today
14, 252 (1993) and U.S. Pat. No. 5,194,594.
Suitable therapeutic agents for forming immunoconjugates of the
present invention include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydro-testosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin, antimetabolites (such as
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, cladribine), alkylating agents (such as
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,
mitomycin C, cisplatin and other platinum derivatives, such as
carboplatin), antibiotics (such as dactinomycin (formerly
actinomycin), bleomycin, daunorubicin (formerly daunomycin),
doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone,
plicamycin, anthramycin (AMC)), diphtheria toxin and related
molecules (such as diphtheria A chain and active fragments thereof
and hybrid molecules), ricin toxin (such as ricin A or a
deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like
toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin,
pertussis toxin, tetanus toxin, soybean Bowman-Birk protease
inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin,
gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin toxins, calicheamicins and
duocarmycins. Therapeutic agents, which may be administered in
combination with a an anti-CD38 antibody of the present invention
as described elsewhere herein, may also be candidates for
therapeutic moieties useful for conjugation to an anti-CD38
antibody of the present invention.
As indicated above, the drug moiety need not be construed as
limited to classical chemical therapeutic agents. For example, the
drug moiety may be a protein or polypeptide possessing a desired
biological activity. In one embodiment, the anti-CD38 antibody of
the present invention is attached to a chelator linker, e.g.
tiuxetan, which allows for the antibody to be conjugated to a
radioisotope.
Bispecific Antibodies
In a further aspect, the invention relates to a bispecific molecule
comprising a first antigen binding site from an anti-CD38 antibody
of the invention as described herein above and a second antigen
binding site with a different binding specificity, such as a
binding specificity for a human effector cell, a human Fc receptor,
a T cell receptor, a B cell receptor or a binding specificity for a
non-overlapping epitope of CD38, i.e. a bispecific antibody wherein
the first and second antigen binding sites do not cross-block each
other for binding to CD38, e.g. when tested as described in Example
3.
Exemplary bispecific antibody molecules of the invention comprise
(i) two antibodies, one with a specificity to CD38 and another to a
second target that are conjugated together, (ii) a single antibody
that has one chain or arm specific to CD38 and a second chain or
arm specific to a second molecule, (iii) a single chain antibody
that has specificity to CD38 and a second molecule, e.g., via two
scFvs linked in tandem by an extra peptide linker; (iv) a
dual-variable-domain antibody (DVD-Ig), where each light chain and
heavy chain contains two variable domains in tandem through a short
peptide linkage (Wu et al., Generation and Characterization of a
Dual Variable Domain Immunoglobulin (DVD-Ig.TM.) Molecule, In:
Antibody Engineering, Springer Berlin Heidelberg (2010)); (v) a
chemically-linked bispecific (Fab').sub.2 fragment; (vi) a Tandab,
which is a fusion of two single chain diabodies resulting in a
tetravalent bispecific antibody that has two binding sites for each
of the target antigens; (vii) a flexibody, which is a combination
of scFvs with a diabody resulting in a multivalent molecule; (viii)
a so called "dock and lock" molecule, based on the "dimerization
and docking domain" in Protein Kinase A, which, when applied to
Fabs, can yield a trivalent bispecific binding protein consisting
of two identical Fab fragments linked to a different Fab fragment;
(ix) a so-called Scorpion molecule, comprising, e.g., two scFvs
fused to both termini of a human Fc-region; and (x) a diabody. In
one embodiment, the bispecific antibody of the present invention is
a diabody, a cross-body, or a bispecific obtained via a controlled
Fab arm exchange as those described in the present invention.
Examples of platforms useful for preparing bispecific antibodies
include but are not limited to BITE (Micromet), DART (MacroGenics),
Fcab and Mab.sup.2 (F-star) , Fc-engineered IgG1 (Xencor) or
DuoBody (based on Fab arm exchange, Genmab, this application).
Examples of different classes of bispecific antibodies include but
are not limited to asymmetric IgG-like molecules, wherein the one
side of the molecule contains the Fab region or part of the Fab
region of at least one antibody, and the other side of the molecule
contains the Fab region or parts of the Fab region of at least one
other antibody; in this class, asymmetry in the Fc region could
also be present, and be used for specific linkage of the two parts
of the molecule; symmetric IgG-like molecules, wherein the two
sides of the molecule each contain the Fab region or part of the
Fab region of at least two different antibodies; IgG fusion
molecules, wherein full length IgG antibodies are fused to extra
Fab regions or parts of Fab regions; Fc fusion molecules, wherein
single chain Fv molecules or stabilized diabodies are fused to
Fc.gamma. regions or parts thereof; Fab fusion molecules, wherein
different Fab-fragments are fused together; ScFv-and diabody-based
molecules wherein different single chain Fv molecules or different
diabodies are fused to eachother or to another protein or carrier
molecule.
Examples of asymmetric IgG-like molecules include but are not
limited to the Triomab/Quadroma (Trion Pharma/Fresenius Biotech),
the Knobs-into-Holes (Genentech), CrossMAbs (Roche) and the
electrostatically-matched (Amgen), the LUZ-Y (Genentech), the
Strand Exchange Engineered Domain body (EMD Serono), the Biclonic
(Merus) and the DuoBody (Genmab A/S).
Example of symmetric IgG-like molecules include but are not limited
to Dual Targeting (DT)-Ig (GSK/Domantis), Two-in-one Antibody
(Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb.sup.2
(F-Star) and CovX-body (CovX/Pfizer).
Examples of IgG fusion molecules include but are not limited to
Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific
(ImClone/Eli Lilly), Ts2Ab (MedImmune/AZ) and BsAb (Zymogenetics),
HERCULES (Biogen Idec) and TvAb (Roche).
Examples of Fc fusion molecules include but are not limited to
ScFv/Fc Fusions (Academic Institution), SCORPION (Emergent
BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting
Technology (Fc-DART) (MacroGenics) and Dual(ScFv).sub.2-Fab
(National Research Center for Antibody Medicine--China).
Examples of class V bispecific antibodies include but are not
limited to F(ab).sub.2 (Medarex/Amgen), Dual-Action or Bis-Fab
(Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent
Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech). Examples of
ScFv-and diabody-based molecules include but are not limited to
Bispecific T Cell Engager (BITE) (Micromet9, Tandem Diabody
(Tandab) (Affimed), Dual Affinity Retargeting Technology (DART)
(MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies
(AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack)
and COMBODY (Epigen Biotech).
In a further aspect, the invention relates to a bispecific molecule
comprising an anti-CD38 antibody of the invention as described
herein above and a second binding specificity such as a binding
specificity for human cytokines. In one embodiment, said cytokine
is an anti-inflammatory cytokine such as IL-1ra, IL-4, IL-6, IL-10,
IL-11, IL-13, IL-16, IFN-alpha and TGF-beta. In another embodiment
said cytokine is a pro-inflammatory cytokine such as IL-lalpha,
IL-ibeta and IL-6. In an embodiment the binding specificity is for
a human effector cell, a human Fc receptor or a T cell receptor. In
one embodiment, said T cell receptor is CD3. In another embodiment,
said human Fc receptor is human Fc.gamma.RI (CD64), human
Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16) or a human Fc.alpha.
receptor (CD89). Bispecific molecules of the present invention may
further include a third binding specificity, in addition to an
anti-CD38 binding specificity and a binding specificity for a human
effector cell, a human Fc receptor or a T cell receptor.
Exemplary bispecific antibody molecules of the invention comprise
(i) two antibodies one with a specificity to CD38 and another to a
second target that are conjugated together, (ii) a single antibody
that has one chain specific to CD38 and a second chain specific to
a second molecule, and (iii) a single chain antibody that has
specificity to CD38 and a second molecule. In one embodiment, the
second molecule is a cancer antigen/tumor-associated antigen such
as CD20, carcinoembryonic antigen (CEA), prostate specific antigen
(PSA), RAGE (renal antigen), a-fetoprotein, CAMEL (CTL-recognized
antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3,
and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE),
mucin antigens (e.g., MUC1, mucin-CA125, etc.), ganglioside
antigens, tyrosinase, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2,
MUM-3, HLA-B7, and Ep-CAM. In one embodiment, the second molecule
is a cancer-associated integrin, such as .alpha.5.beta.3 integrin.
In one embodiment, the second molecule is an angiogenic factor or
other cancer-associated growth factor, such as a vascular
endothelial growth factor (VEGF), a fibroblast growth factor (FGF),
epidermal growth factor (EGF), epidermal growth factor receptor
(EGFR), angiogenin, and receptors thereof, particularly receptors
associated with cancer progression (for instance one of the
HER1-HER4 receptors). Other cancer progression-associated proteins
discussed herein may also be suitable second molecules.
In an embodiment of the invention, the antibody is a single
antibody that has one chain specific to the CD38 epitope described
in this invention comprising aspartic acid at position 202 and a
second chain specific for a CD38 specific epitope that does not
comprise the aspartic acid at position 202 (a non-competing
antibody). Such antibody is described for example in WO2006099875
as antibody 003.
In one embodiment, a bispecific antibody of the present invention
is a diabody.
Generation of Bispecific Antibodies by 2-MEA-induced Fab-Arm
Exchange
An in vitro method for producing bispecific antibodies is described
in WO 2008119353 (Genmab) and reported by van der Neut-Kolfschoten
et al. (Science. 2007 Sep 14;317(5844):1554-7). Herein, a
bispecific antibody is formed by "Fab-arm" or "half-molecule"
exchange (swapping of a heavy chain and attached light chain)
between two monospecific IgG4-or IgG4-like antibodies upon
incubation under mildly reducing conditions. This Fab-arm exchange
reaction is the result of a disulfide-bond isomerization reaction
wherein the inter heavy-chain disulfide bonds in the hinge regions
of monospecific antibodies are reduced and the resulting free
cysteines form a new inter heavy-chain disulfide bond with cysteine
residues of another antibody molecule with a different specificity.
The resulting product is a bispecific antibody having two Fab arms
with different sequences.
In a novel invention the knowledge of this natural IgG4 Fab-arm
exchange is adapted to generate a method to produce stable
IgG1-based bispecific antibodies. The bispecific antibody product
generated by this method described below will no longer participate
in IgG4 Fab-arm exchange. The basis for this method is the use of
complimentary CH3 domains, which promote the formation of
heterodimers under specific assay conditions. To enable the
production of bispecific antibodies by this method, IgG1 molecules
carrying certain mutations in the CH3 domain were generated: in one
of the parental IgG1 antibody T350I, K370T and F405L mutations in
the other parental IgG1 antibody the K409R mutation.
To generate bispecific antibodies, these two parental antibodies,
each antibody at a final concentration of 0.5 mg/mL (equimolar
concentration), were incubated with 25 mM 2-mercaptoethylamine-HCl
(2-MEA) in a total volume of 100 .mu.L TE at 37.degree. C. for 90
min. The reduction reaction is stopped when the reducing agent
2-MEA is removed by using spin columns (Microcon centrifugal
filters, 30 k, Millipore) according to the manufacturer's protocol.
By this method the following bispecific antibodies may be
generated:
A bispecfic antibody wherein the anti-CD38 antibody is 025, 026,
028, 049 or 056, and the second binding moiety is an anti-CD3
antibody.
A bispecfic antibody wherein the anti-CD38 antibody is 025, 026,
028, 049 or 056, and the second binding moiety is an anti-CD20
antibody, such as ofatumumab.
A bispecfic antibody wherein the anti-CD38 antibody is 025, 026,
028, 049 or 056, and the second binding moiety is an anti-CD16
antibody.
A bispecfic antibody wherein the anti-CD38 antibody is 025, 026,
028, 049 or 056, and the second binding moiety is an anti-CD32
antibody.
A bispecfic antibody wherein the anti-CD38 antibody is 025, 026,
028, 049 or 056, and the second binding moiety is an anti-CD64
antibody.
Nucleic Acids, Vectors, Host Cells and Method for Producing
Antibodies of the Invention
In a further aspect, the invention relates to nucleic acids
encoding (parts of) an antibody of the invention and to expression
vectors comprising such nucleic acids.
In one embodiment, the expression vector of the invention comprises
a nucleotide sequence encoding one or more of the amino acid
sequences selected from the group consisting of: SEQ ID NO: 1 and
SEQ ID NO: 5.
In a further embodiment, the expression vector further comprises a
nucleotide sequence encoding the constant region of a light chain,
a heavy chain or both light and heavy chains of an antibody, e.g. a
human antibody.
Such expression vectors may be used for recombinant production of
antibodies of the invention.
An expression vector in the context of the present invention may be
any suitable vector, including chromosomal, non-chromosomal, and
synthetic nucleic acid vectors (a nucleic acid sequence comprising
a suitable set of expression control elements). Examples of such
vectors include derivatives of SV40, bacterial plasmids, phage DNA,
baculovirus, yeast plasmids, vectors derived from combinations of
plasmids and phage DNA, and viral nucleic acid (RNA or DNA)
vectors. In one embodiment, an anti-CD38 antibody-encoding nucleic
acid is comprised in a naked DNA or RNA vector, including, for
example, a linear expression element (as described in for instance
Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted
nucleic acid vector (as described in for instance U.S. Pat. No.
6,077,835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC
19/18, or pUC 118/119, a "midge" minimally-sized nucleic acid
vector (as described in for instance Schakowski et al., Mol Ther 3,
793-800 (2001)), or as a precipitated nucleic acid vector
construct, such as a CaPO4-precipitated construct (as described in
for instance WO 00/46147, Benvenisty and Reshef, PNAS USA 83,
9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and
Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid
vectors and the usage thereof are well known in the art (see for
instance U.S. Pat. Nos. 5,589,466 and 5,973,972).
In one embodiment, the vector is suitable for expression of the
anti-CD38 antibody in a bacterial cell. In another embodiment, the
expression vector may be a vector suitable for expression in a
yeast system. Most typically, the vector will be a vector suitable
for expression of the antibody of the invention in a mammalian
cell, such as a CHO, HEK or PER.C6.RTM. cell (human cell line
developed by DSM and Crucell N.V., the Netherlands). Another
suitable vector system is the glutamine synthetase (GS) vector
system developed by Lonza Biologics (see e.g. EP216846, U.S. Pat.
No. 5,981,216, WO8704462, EP323997, U.S. Pat. Nos. 5,591,639,
5,658,759, EP338841, U.S. Pat. Nos. 5,879,936, and 5,891,693).
In an expression vector of the invention, anti-CD38
antibody-encoding nucleic acids may comprise or be associated with
any suitable promoter, enhancer, and other expression-facilitating
elements. Examples of such elements include strong expression
promoters (e. g., human CMV IE promoter/enhancer as well as RSV,
SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A)
termination sequences, an origin of replication for plasmid product
in E. coli, an antibiotic resistance gene as selectable marker,
and/or a convenient cloning site (e.g., a polylinker). Nucleic
acids may also comprise an inducible promoter as opposed to a
constitutive promoter such as CMV IE.
In one embodiment, the anti-CD38-antibody-encoding expression
vector may be positioned in and/or delivered to the host cell or
host animal via a viral vector.
In an even further aspect, the invention relates to a recombinant
eukaryotic or prokaryotic host cell, such as a transfectoma, which
produces an antibody of the invention as defined herein. Examples
of host cells include yeast, bacterial, and mammalian cells, such
as a CHO, HEK or PER.C6.RTM. cells. For example, in one embodiment,
the present invention provides a cell comprising a nucleic acid
stably integrated into the cellular genome that comprises a
sequence coding for expression of an anti-CD38 antibody of the
present invention. In another embodiment, the present invention
provides a cell comprising a non-integrated nucleic acid, such as a
plasmid, cosmid, phagemid, or linear expression element, which
comprises a sequence coding for expression of an anti-CD38 antibody
of the invention.
In a further aspect, the invention relates to a hybridoma which
produces an antibody of the invention as defined herein. In an even
further aspect, the invention relates to a transgenic non-human
animal comprising nucleic acids encoding a human heavy chain and a
human light chain, wherein the animal or plant produces an antibody
of the invention. Generation of such hybridomas and transgenic
animals has been described above.
In a further aspect, the invention relates to amethod for producing
an anti-CD38 antibody of the invention, said method comprising the
steps of: a) culturing a hybridoma or a host cell of the invention
as described herein above, and b) purifying the antibody of the
invention from the culture media. Pharmaceutical Compositions
In an even further aspect, the invention relates to a
pharmaceutical composition comprising: an anti-CD38 antibody as
defined herein, and a pharmaceutically-acceptable carrier.
The pharmaceutical compositions may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques such as those disclosed in Remington: The
Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack
Publishing Co., Easton, Pa., 1995. A pharmaceutical composition of
the present invention may also include diluents, fillers, salts,
buffers, detergents (e. g., a nonionic detergent, such as Tween-20
or Tween-80), stabilizers (e. g., sugars or protein-free amino
acids), preservatives, tissue fixatives, solubilizers, and/or other
materials suitable for inclusion in a pharmaceutical
composition.
The pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients should be suitable for the
chosen compound of the present invention and the chosen mode of
administration. Suitability for carriers and other components of
pharmaceutical compositions is determined based on the lack of
significant negative impact on the desired biological properties of
the chosen compound or pharmaceutical composition of the present
invention (e.g., less than a substantial impact (10% or less
relative inhibition, 5% or less relative inhibition, etc.)) on
antigen binding.
The actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the amide thereof, the route of administration, the
time of administration, the rate of excretion of the particular
compound being employed, the duration of the treatment, other
drugs, compounds and/or materials used in combination with the
particular compositions employed, the age, sex, weight, condition,
general health and prior medical history of the patient being
treated, and like factors well known in the medical arts.
The pharmaceutical composition may be administered by any suitable
route and mode. Suitable routes of administering a compound of the
present invention in vivo and in vitro are well known in the art
and may be selected by those of ordinary skill in the art.
In one embodiment, a pharmaceutical composition of the present
invention is administered parenterally.
The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
include epidermal, intravenous, intramuscular, intraarterial,
intrathecal, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, intratendinous, transtracheal,
subcutaneous, subcuticular, intraarticular, subcapsular,
subarachnoid, intraspinal, intracranial, intrathoracic, epidural
and intrasternal injection and infusion.
In one embodiment that pharmaceutical composition is administered
by intravenous or subcutaneous injection or infusion.
In one embodiment the compounds of the present invention are
administered in crystalline form by subcutaneous injection, cf.
Yang et al., PNAS USA 100(12), 6934-6939 (2003).
Pharmaceutically acceptable carriers include any and all suitable
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonicity agents, antioxidants and absorption delaying
agents, and the like that are physiologically compatible with a
compound of the present invention.
Examples of suitable aqueous and nonaqueous carriers which may be
employed in the pharmaceutical compositions of the present
invention include water, saline, phosphate buffered saline,
ethanol, dextrose, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), and suitable mixtures thereof,
vegetable oils, carboxymethyl cellulose colloidal solutions,
tragacanth gum and injectable organic esters, such as ethyl oleate,
and/or various buffers. Other carriers are well known in the
pharmaceutical arts.
Pharmaceutical compositions of the present invention may also
comprise pharmaceutically acceptable antioxidants for instance (1)
water soluble antioxidants, such as ascorbic acid, cysteine
hydrochloride, sodium bisulfate, sodium metabisulfite, sodium
sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, and the like.
Pharmaceutical compositions of the present invention may also
comprise isotonicity agents, such as sugars, polyalcohols, such as
mannitol, sorbitol, glycerol or sodium chloride.
The pharmaceutical compositions of the present invention may also
contain one or more adjuvants appropriate for the chosen route of
administration such as preservatives, wetting agents, emulsifying
agents, dispersing agents, preservatives or buffers, which may
enhance the shelf life or effectiveness of the pharmaceutical
composition. The compounds of the present invention may be prepared
with carriers that will protect the compound against rapid release,
such as a controlled release formulation, including implants,
transdermal patches, and microencapsulated delivery systems.
Methods for the preparation of such formulations are generally
known to those skilled in the art. See e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978.
The pharmaceutical composition of the present invention may contain
one antibody of the present invention or a combination of two or
more antibodies of the present invention.
Therapeutic Uses
In another aspect, the invention relates to the antibody of the
invention as defined herein for use as a medicament.
The anti-CD38 antibodies of the present invention have numerous
therapeutic utilities involving the treatment of disorders
involving cells expressing CD38. For example, the antibodies may be
administered to cells in culture, e.g., in vitro or ex vivo, or to
human subjects, e.g., in vivo, to treat or prevent a variety of
disorders. As used herein, the term "subject" is intended to
include human and non-human animals which respond to the antibody.
Subjects may for instance include human patients having disorders
that may be corrected or ameliorated by modulating CD38 function,
such as enzymatic activity, signal transduction, induction of
cytokine expression, induction of proliferation or differentiation,
and/or induction of lysis and/or eliminating/reducing the number of
CD38 expressing cells.
For example, the anti-CD38 antibodies may be used to elicit in vivo
or in vitro one or more of the following biological activities:
modulating CD38 function (such as enzymatic activity, signal
transduction, induction of cytokine expression, induction of
proliferation or differentiation, and/or induction of lysis),
killing a cell expressing CD38, mediating phagocytosis or ADCC of a
cell expressing CD38 in the presence of human effector cells, and
by mediating CDC of a cell expressing CD38 in the presence of
complement or by killing CD38 expressing cells by apoptosis.
The present invention provides methods for treating or preventing a
disorder involving cells expressing CD38 in a subject, which method
comprises administration of a therapeutically effective amount of
an anti-CD38 antibody of the present invention to a subject in need
thereof. Such a method involves administering to a subject an
anti-CD38 antibody of the present invention in an amount effective
to treat or prevent the disorder.
In one embodiment of the present invention, the disorder involving
cells expressing CD38 may be cancer, i.e. a tumorigenic disorder,
such as a disorder characterized by the presence of tumor cells
expressing CD38 including, for example, B cell lymphoma, plasma
cell malignancies, T/NK cell lymphoma and myeloid malignancies.
Examples of such tumorigenic diseases include B cell
lymphomas/leukemias including precursor B cell lymphoblastic
leukemia/lymphoma and B cell non-Hodgkin's lymphomas; acute
promyelocytic leukemia, acute lymphoblastic leukemia and mature B
cell neoplasms, such as B cell chronic lymhocytic
leukemia(CLL)/small lymphocytic lymphoma (SLL), B cell acute
lymphocytic leukemia, B cell prolymphocytic leukemia,
lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular
lymphoma (FL), including low-grade, intermediate-grade and
high-grade FL, cutaneous follicle center lymphoma, marginal zone B
cell lymphoma (MALT type, nodal and splenic type), hairy cell
leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma,
plasmacytoma, plasma cell myeloma, plasma cell leukemia,
post-transplant lymphoproliferative disorder, Waldenstrom's
macroglobulinemia, plasma cell leukemias and anaplastic large-cell
lymphoma (ALCL).
In one embodiment, the disorder involving cells expressing CD38 is
multiple myeloma.
In one embodiment, the disorder involving cells expressing CD38 is
selected from chronic lymphocytic leukemia (CLL), acute
lymphoblastic leukemia (ALL), acute myelogenous leukemia (adults)
(AML), mantle cell lymphoma, follicular lymphoma, and diffuse large
B-cell lymphoma.
In one embodiment the disorder involving cells expressing CD38 is
non-small cell lung cancer (NSCLC).
Examples of B cell non-Hodgkin's lymphomas are lymphomatoid
granulomatosis, primary effusion lymphoma, intravascular large B
cell lymphoma, mediastinal large B cell lymphoma, heavy chain
diseases (including .gamma., .mu., and .alpha. disease), lymphomas
induced by therapy with immunosuppressive agents, such as
cyclosporine-induced lymphoma, and methotrexate-induced
lymphoma.
In one embodiment of the present invention, the disorder involving
cells expressing CD38 is Hodgkin's lymphoma.
Other examples of disorders involving cells expressing CD38 include
malignancies derived from T and NK cells including: mature T cell
and NK cell neoplasms including T cell prolymphocytic leukemia, T
cell large granular lymphocytic leukemia, aggressive NK cell
leukemia, adult T cell leukemia/lymphoma, extranodal NK/T cell
lymphoma, nasal type, enteropathy-type T cell lymphoma,
hepatosplenic T cell lymphoma, subcutaneous panniculitis-like T
cell lymphoma, blastic NK cell lymphoma, Mycosis Fungoides/Sezary
Syndrome, primary cutaneous CD30 positive T cell
lymphoproliferative disorders (primary cutaneous anaplastic large
cell lymphoma C-ALCL, lymphomatoid papulosis, borderline lesions),
angioimmunoblastic T cell lymphoma, peripheral T cell lymphoma
unspecified, and anaplastic large cell lymphoma.
Examples of malignancies derived from myeloid cells include acute
myeloid leukemia, including acute promyelocytic leukemia, and
chronic myeloproliferative diseases, including chronic myeloid
leukemia.
In another embodiment of the present invention, the disorder
involving cells expressing CD38 is an immune disorder in which CD38
expressing B cells, macrophages, plasma cells, monocytes and T
cells are involved, such as an inflammatory and/or autoimmune
disease. Examples of immune disorders in which CD38 expressing B
cells, plasma cells, monocytes and T cells are involved include
autoimmune disorders, such as psoriasis, psoriatic arthritis,
dermatitis, systemic scleroderma and sclerosis, inflammatory bowel
disease (IBD), Crohn's disease, ulcerative colitis, respiratory
distress syndrome, meningitis, encephalitis, uveitis,
glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte
adhesion deficiency, multiple sclerosis, Raynaud's syndrome,
Sjogren's syndrome, juvenile onset diabetes, Reiter's disease,
Behcet's disease, immune complex nephritis, IgA nephropathy, IgM
polyneuropathies, immune-mediated thrombocytopenias, such as acute
idiopathic thrombocytopenic purpura and chronic idiopathic
thrombocytopenic purpura, hemolytic anemia, myasthenia gravis,
lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis
(RA), atopic dermatitis, pemphigus, Graves' disease, Hashimoto's
thyroiditis, Wegener's granulomatosis, Omenn's syndrome, chronic
renal failure, acute infectious mononucleosis, multiple sclerosis,
HIV, and herpes virus associated diseases. Further examples are
severe acute respiratory distress syndrome and choreoretinitis.
Furthermore, other diseases and disorders are included such as
those caused by or mediated by infection of B-cells with virus,
such as Epstein-Barr virus (EBV).
In one embodiment, the disorder involving cells expressing CD38 is
rheumatoid arthritis.
Further examples of inflammatory, immune and/or autoimmune
disorders in which autoantibodies and/or excessive B and T
lymphocyte activity are prominent and which may be treated
according to the present invention include the following:
vasculitides and other vessel disorders, such as microscopic
polyangiitis, Churg-Strauss syndrome, and other ANCA-associated
vasculitides, polyarteritis nodosa, essential cryoglobulinaemic
vasculitis, cutaneous leukocytoclastic angiitis, Kawasaki disease,
Takayasu arteritis, giant cell arthritis, Henoch-Schonlein purpura,
primary or isolated cerebral angiitis, erythema nodosum,
thrombangiitis obliterans, thrombotic thrombocytopenic purpura
(including hemolytic uremic syndrome), and secondary vasculitides,
including cutaneous leukocytoclastic vasculitis (e.g., secondary to
hepatitis B, hepatitis C, Waldenstrom's macroglobulinemia, B-cell
neoplasias, rheumatoid arthritis, Sjogren's syndrome, or systemic
lupus erythematosus); further examples are erythema nodosum,
allergic vasculitis, panniculitis, Weber-Christian disease, purpura
hyperglobulinaemica, and Buerger's disease; skin disorders, such as
contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma
gangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris
(including cicatricial pemphigoid and bullous pemphigoid), alopecia
areata (including alopecia universalis and alopecia totalis),
dermatitis herpetiformis, erythema multiforme, and chronic
autoimmune urticaria (including angioneurotic edema and urticarial
vasculitis); immune-mediated cytopenias, such as autoimmune
neutropenia, and pure red cell aplasia; connective tissue
disorders, such as CNS lupus, discoid lupus erythematosus, CREST
syndrome, mixed connective tissue disease,
polymyositis/dermatomyositis, inclusion body myositis, secondary
amyloidosis, cryoglobulinemia type I and type II, fibromyalgia,
phospholipid antibody syndrome, secondary hemophilia, relapsing
polychondritis, sarcoidosis, stiff man syndrome, and rheumatic
fever; a further example is eosinophil fasciitis; arthritides, such
as ankylosing spondylitis, juvenile chronic arthritis, adult
Still's disease, and SAPHO syndrome; further examples are
sacroileitis, reactive arthritis, Still's disease, and gout;
hematologic disorders, such as aplastic anemia, primary hemolytic
anemia (including cold agglutinin syndrome), hemolytic anemia
secondary to CLL or systemic lupus erythematosus; POEMS syndrome,
pernicious anemia, and Waldemstrom's purpura hyperglobulinaemica;
further examples are agranulocytosis, autoimmune neutropenia,
Franklin's disease, Seligmann's disease, gamma heavy chain disease,
paraneoplastic syndrome secondary to thymoma and lymphomas, an,
paraneoplastic syndrome secondary to thymoma and lymphomas, and
factor VIII inhibitor formation; endocrinopathies, such as
polyendocrinopathy, and Addison's disease; further examples are
autoimmune hypoglycemia, autoimmune hypothyroidism, autoimmune
insulin syndrome, de Quervain's thyroiditis, and insulin receptor
antibody-mediated insulin resistance; hepato-gastrointestinal
disorders, such as celiac disease, Whipple's disease, primary
biliary cirrhosis, chronic active hepatitis, and primary sclerosing
cholangiitis; a further example is autoimmune gastritis;
nephropathies, such as rapid progressive glomerulonephritis,
post-streptococcal nephritis, Goodpasture's syndrome, membranous
glomerulonephritis, and cryoglobulinemic nephritis; a further
example is minimal change disease; neurological disorders, such as
autoimmune neuropathies, mononeuritis multiplex, Lambert-Eaton's
myasthenic syndrome, Sydenham's chorea, tabes dorsalis, and
Guillain-Barre's syndrome; further examples are myelopathy/tropical
spastic paraparesis, myasthenia gravis, acute inflammatory
demyelinating polyneuropathy, and chronic inflammatory
demyelinating polyneuropathy; multiple sclerosis; cardiac and
pulmonary disorders, such as COPD, fibrosing alveolitis,
bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis,
Loffler's syndrome, myocarditis, and pericarditis; further examples
are hypersensitivity pneumonitis, and paraneoplastic syndrome
secondary to lung cancer; allergic disorders, such as bronchial
asthma and hyper-IgE syndrome; a further example is amaurosis
fugax; ophthalmologic disorders, such as idiopathic
chorioretinitis; infectious diseases, such as parvovirus B
infection (including hands-and-socks syndrome);
gynecological-obstretical disorders, such as recurrent abortion,
recurrent fetal loss, and intrauterine growth retardation; a
further example is paraneoplastic syndrome secondary to
gynaecological neoplasms; male reproductive disorders, such as
paraneoplastic syndrome secondary to testicular neoplasms; and
transplantation-derived disorders, such as allograft and xenograft
rejection, and graft-versus-host disease.
Dosage regimens in the above methods of treatment and uses are
adjusted to provide the optimum desired response (e.g., a
therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation.
The efficient dosages and the dosage regimens for the anti-CD38
antibodies depend on the disease or condition to be treated and may
be determined by the persons skilled in the art. An exemplary,
non-limiting range for a therapeutically effective amount of a
compound of the present invention is about 0.005-100 mg/kg, such as
0.05-100 mg/kg or 1-100 mg/kg, such as about 0.1-50 mg/kg, for
example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for
instance about 0.1, 0.3, about 0.5, about 1, 2, 3, 4, 8, 16 or 24
mg/kg.
Administration may e.g. be intravenous, intramuscular,
intraperitoneal, or subcutaneous, and for instance administered
proximal to the site of the target. If desired, the effective daily
dose of a pharmaceutical composition may be administered as two,
three, four, five, six or more sub-doses administered separately at
appropriate intervals throughout the day, optionally, in unit
dosage forms.
In one embodiment, the anti-CD38 antibodies may be administered by
infusion in a weekly dosage of from 10 to 500 mg/m.sup.2, such as
of from 200 to 400 mg/m.sup.2. Such administration may be repeated,
e.g., 1 to 8 times, such as 3 to 5 times. The administration may be
performed by continuous infusion over a period of from 2 to 24
hours, such as of from 2 to 12 hours.
In one embodiment, the anti-CD38 antibodies may be administered by
slow continuous infusion over a long period, such as more than 24
hours, in order to reduce toxic side effects.
In one embodiment the anti-CD38 antibodies may be administered in a
weekly dosage of from 250 mg to 2000 mg, such as for example 300
mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times,
such as from 4 to 6 times. The administration may be performed by
continuous infusion over a period of from 2 to 24 hours, such as of
from 2 to 12 hours. Such regimen may e.g. be repeated one or more
times as necessary, for example, after 6 months or 12 months.
In one embodiment, the anti-CD38 antibodies may be administered by
maintenance therapy, such as, e.g., once a week for a period of 6
months or more.
In another embodiment, the anti-CD38 antibodies may be administered
by a regimen including one infusion of an anti-CD38 antibody of the
present invention followed by an infusion of an anti-CD38 antibody
of the present invention conjugated to a radioisotope. The regimen
may be repeated, e.g., 7 to 9 days later.
As non-limiting examples, treatment according to the present
invention may be provided as a daily dosage of a compound of the
present invention in an amount of about 0.1-100 mg/kg, such as 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after
initiation of treatment, or any combination thereof, using single
or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any
combination thereof.
An "effective amount" for tumor therapy may also be measured by its
ability to stabilize the progression of disease. The ability of a
compound to inhibit cancer may be evaluated in an animal model
system predictive of efficacy in human tumors. Alternatively, this
property of a composition may be evaluated by examining the ability
of the compound to inhibit cell growth or to induce apoptosis by in
vitro assays known to the skilled practitioner. A therapeutically
effective amount of a therapeutic compound may decrease tumor size,
or otherwise ameliorate symptoms in a subject.
A "therapeutically effective amount" for rheumatoid arthritis may
result in an at least ACR.sub.20 Preliminary Definition of
Improvement in the patients, such as in at least an ACR.sub.50
Preliminary Definition of Improvement, for instance at least an
ARC.sub.70 Preliminary Definition of Improvement.
ACR.sub.20 Preliminary Definition of Improvement is defined as:
.gtoreq.20% improvement in: Tender Joint Count (TJC) and Swollen
Joint Count (SJC) and .gtoreq.20% improvement in 3 of following 5
assessments: Patient Pain Assessment (VAS), Patient Global
assessment (VAS), Physician Global Assessment (VAS), Patent
Self-Assessed Disability (HAQ), Acute Phase Reactant (CRP or ESR).
ACR.sub.50 and ACR.sub.70 are defined in the same way with
.gtoreq.50% and .gtoreq.70% improvements, respectively. For further
details see Felson et al., in American College of Rheumatology
Preliminary Definition of Improvement in Rheumatoid Arthritis;
Arthritis Rheumatism 38, 727-735 (1995).
Alternatively, a therapeutically effective amount for rheumatoid
arthritis can be measured by DAS (disease activity score),
including DAS28 and/or DAS56, as defined by EU LAR.
An anti-CD38 antibody may also be administered prophylactically in
order to reduce the risk of developing cancer, delay the onset of
the occurrence of an event in cancer progression, and/or reduce the
risk of recurrence when a cancer is in remission. This may be
especially useful in patients wherein it is difficult to locate a
tumor that is known to be present due to other biological
factors.
Combination Therapy
The anti-CD38 antibodies of the present invention may also be
administered in combination therapy, i.e., combined with other
therapeutic agents relevant for the disease or condition to be
treated. Such administration may be simultaneous, separate or
sequential. For simultaneous administration the agents may be
administered as one compositons or as separate compositions, as
appropriate.
Accordingly, the present invention provides methods for treating a
disorder involving cells expressing CD38 as described above, which
methods comprise administration of an anti-CD38 antibody of the
present invention combined with one or more additional therapeutic
agents as described below.
In an embodiment of the invention the antibodies of the present
invention are administered as a combination with other anti-CD38
antibodies. Such antibodies are described in the present invention
and in prior art. Specifically, antibodies are described in
WO2006099875. More specifically, a combination of the present
anti-CD38 antibodies with anti-CD38 antibodies which are
non-cross-blocking, such as antibody 003 described in WO2006099875
are embodiments of the present invention.
In one embodiment, the combination therapy may include
administration of a composition of the present invention together
with at least one cytotoxic agent, at least one chemotherapeutic
agent, at least one anti-angiogenic agent, at least one
anti-inflammatory agent, and/or at least one immunosuppressive
and/or immunomodulatory agent.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38, such as
cancer, in a subject, which method comprises administration of a
therapeutically effective amount of an anti-CD38 antibody of the
present invention and at least one chemotherapeutic agent to a
subject in need thereof
In one embodiment, the present invention provides a method for
treating multiple myeloma, which method comprises administration of
a therapeutically effective amount of an anti-CD38 antibody of the
present invention and at least one chemotherapeutic agent to a
subject in need thereof.
In one embodiment, such a chemotherapeutic agent may be selected
from an antimetabolite, such as methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, fludarabine, 5-fluorouracil,
decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine and
similar agents.
In one embodiment, such a chemotherapeutic agent may be selected
from an alkylating agent, such as mechlorethamine, thioepa,
chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),
cyclophosphamide, busulfan, dibromomannitol, streptozotocin,
dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other
platinum derivatives, such as carboplatin, and similar agents.
In one embodiment, such a chemotherapeutic agent may be selected
from an antibiotic, such as dactinomycin (formerly actinomycin),
bleomycin, daunorubicin (formerly daunomycin), doxorubicin,
idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin,
anthramycin (AMC) and similar agents.
In one embodiment, such a chemotherapeutic agent may be selected
from an anti-mitotic agent, such as taxanes, for instance
docetaxel, and paclitaxel, and vinca alkaloids, for instance
vindesine, vincristine, vinblastine, and vinorelbine.
In one embodiment, such a chemotherapeutic agent may be selected
from a topoisomerase inhibitor, such as topotecan.
In one embodiment, such a chemotherapeutic agent may be selected
from a growth factor inhibitor, such as an inhibitor of ErbB1
(EGFR) (such as gefitinib (Iressa.RTM.), cetuximab (Erbitux.RTM.),
erlotinib (Tarceva.RTM.), HuMax-EGFr (zalutumumab, 2F8 disclosed in
WO 2002/100348) and similar agents), an inhibitor of ErbB2
(Her2/neu) (such as trastuzumab (Herceptin.RTM.) and similar
agents) and similar agents. In one embodiment, such a growth factor
inhibitor may be a farnesyl transferase inhibitor, such as
SCH-66336 and R115777. In one embodiment, such a growth factor
inhibitor may be a vascular endothelial growth factor (VEGF)
inhibitor, such as bevacizumab (Avastin.RTM.).
In one embodiment, such a chemotherapeutic agent may be a tyrosine
kinase inhibitor, such as imatinib (Glivec, Gleevec STI571),
lapatinib, PTK787/ZK222584 and similar agents.
In one embodiment, such a chemotherapeutic agent may be a histone
deacetylase inhibitor. Examples of such histone deacetylase
inhibitors include hydroxamic acid-based hybrid polar compounds,
such as SAHA (suberoylanilide hydroxamic acid).
In one embodiment, such a chemotherapeutic agent may be a P38a MAP
kinase inhibitor, such as SCIO-469.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one inhibitor of angiogenesis, neovascularization,
and/or other vascularization to a subject in need thereof
In one embodiment, the present invention provides a method for
treating multiple myeloma, which method comprises administration of
a therapeutically effective amount of an anti-CD38 antibody of the
present invention and at least one inhibitor of angiogenesis,
neovascularization, and/or other vascularization to a subject in
need thereof.
Examples of such angiogenesis inhibitors are urokinase inhibitors,
matrix metalloprotease inhibitors (such as marimastat, neovastat,
BAY 12-9566, AG 3340, BMS-275291 and similar agents), inhibitors of
endothelial cell migration and proliferation (such as TNP-470,
squalamine, 2-methoxyestradiol, combretastatins, endostatin,
angiostatin, penicillamine, SCH66336 (Schering-Plough Corp,
Madison, N.J.), R115777 (Janssen Pharmaceutica, Inc, Titusville,
N.J.) and similar agents), antagonists of angiogenic growth factors
(such as ZD6474, SU6668, antibodies against angiogenic agents
and/or their receptors (such as VEGF, bFGF, and angiopoietin-1),
thalidomide (Thalomid.RTM.), thalidomide analogs (such as CC-5013
(lenalidomide, Revlimid.TM.) and CC4047 (Actimid.TM.), Sugen 5416,
SU5402, antiangiogenic ribozyme (such as angiozyme), interferon a
(such as interferon .alpha.2a), suramin and similar agents), VEGF-R
kinase inhibitors and other anti-angiogenic tyrosine kinase
inhibitors (such as SU011248), inhibitors of endothelial-specific
integrin/survival signaling (such as vitaxin and similar agents),
copper antagonists/chelators (such as tetrathiomolybdate, captopril
and similar agents), carboxyamido-triazole (CAI), ABT-627, CM101,
interleukin-12 (IL-12), IM862, PNU145156E as well as nucleotide
molecules inhibiting angiogenesis (such as antisense-VEGF-cDNA,
cDNA coding for angiostatin, cDNA coding for p53 and cDNA coding
for deficient VEGF receptor-2) and similar agents.
Other examples of such inhibitors of angiogenesis,
neovascularization, and/or other vascularization are
anti-angiogenic heparin derivatives and related molecules (e.g.,
heperinase III), temozolomide, NK4, macrophage migration inhibitory
factor (MIF), cyclooxygenase-2 inhibitors, inhibitors of
hypoxia-inducible factor 1, anti-angiogenic soy isoflavones,
oltipraz, fumagillin and analogs thereof, somatostatin analogues,
pentosan polysulfate, tecogalan sodium, dalteparin, tumstatin,
thrombospondin, NM-3, combrestatin, canstatin, avastatin,
antibodies against other relevant targets (such as
anti-alpha-v/beta-3 integrin and anti-kininostatin mAbs) and
similar agents.
In one embodiment, the present invention provides the use of an
anti-CD38 antibody of the present invention for the preparation of
a pharmaceutical composition to be administered with thalidomide
(Thalomid.RTM.), thalidomide analogs (such as CC-5013
(lenalidomide, Revlimid.TM.) and/or CC4047 (Actimid.TM.). In a
further embodiment, the present invention provides the use of an
anti-CD38 antibody of the present invention for the preparation of
a pharmaceutical composition to be administered with
thalidomide.
In one embodiment, the present invention provides the use of an
anti-CD38 antibody of the present invention for the preparation of
a pharmaceutical composition to be administered with an anti-CD20
antibody, such as rituximab (Rituxan.RTM., Mabthera.RTM.), a human
monoclonal anti-CD20 antibody as disclosed in WO 2004/035607, such
as 11B8, 2F2 (ofatumumab, Arzerra.RTM.) or 7D8.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a proteasome inhibitor, such as
bortezomib (Velcade.RTM.).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a corticosteroid, such as
prednisone, prednisolone, dexamethasone, etc.
In one embodiment, the anti-CD38 antibody of the present invention
is used in combination with lenalidomide and dexamethasone for
treating the disorders as described above, such as multiple
myeloma, e.g. relapsed multiple myeloma.
In one embodiment, the anti-CD38 antibody of the present invention
is used in combination with bortezomib and dexamethasone for
treating the disorders as described above, such as multiple
myeloma, e.g. relapsed multiple myeloma.
In one embodiment, the anti-CD38 antibody of the present invention
is used in combination with bortezomib and prednisolone for
treating the disorders as described above, such as multiple
myeloma, e.g. relapsed multiple myeloma.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be an anti-cancer immunogen, such
as a cancer antigen/tumor-associated antigen (e.g., epithelial cell
adhesion molecule (EpCAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic
antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100,
Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines
(e.g., human papillomavirus vaccines), tumor-derived heat shock
proteins, and similar agents. A number of other suitable cancer
antigens/tumor-associated antigens described elsewhere herein and
similar molecules known in the art may also or alternatively be
used in such embodiment. Anti-cancer immunogenic peptides also
include anti-idiotypic "vaccines" such as BEC2 anti-idiotypic
antibodies, Mitumomab, CeaVac and related anti-idiotypic
antibodies, anti-idiotypic antibody to MG7 antibody, and other
anti-cancer anti-idiotypic antibodies (see for instance Birebent et
al., Vaccine. 21(15), 1601-12 (2003), Li et al., Chin Med J (Eng!).
114(9), 962-6 (2001), Schmitt et al., Hybridoma. 0 (5), 389-96
(1994), Maloney et al., Hybridoma. 4(3), 191-209 (1985),
Raychardhuri et al., J Immunol. 137(5), 1743-9 (1986), Pohl et al.,
Int3 Cancer. 50(6), 958-67 (1992), Bohlen et al., Cytokines Mol
Ther. 2(4), 231-8 (1996) and Maruyama, J Immunol Methods. 264(1-2),
121-33 (2002)). Such anti-idiotypic Antibodies may optionally be
conjugated to a carrier, which may be a synthetic (typically inert)
molecule carrier, a protein (for instance keyhole limpet hemocyanin
(KLH) (see for instance Ochi et al., Eur J Immunol. 17(11), 1645-8
(1987)), or a cell (for instance a red blood cell--see for instance
Wi et al., J Immunol Methods. 122(2), 227-34 (1989)).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a bisphosphonate. Examples of
potentially suitable biphosphonates are pamidronate (Aredia.RTM.),
zoledronic acid (Zometa.RTM.), clodronate (Bonefos.RTM.),
risendronate (Actonel.RTM.), ibandronate (Boniva.RTM.), etidronate
(Didronel.RTM.), alendronate (Fosamax.RTM.), tiludronate
(Skelid.RTM.), incadronate (Yamanouchi Pharmaceutical) and
minodronate (YM529, Yamanouchi).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a colony stimulating factor.
Examples of suitable colony stimulating factors are
granulocyte-colony stimulating factors (G-CSF), such as filgrastim
(Neupogen.RTM.) and pegfilgrastim (Neulasta.RTM.), and granulocyte
macrophage-colony stimulating factors (GM-CSF) such as sargramostim
(Leukine.RTM.).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a erythropoietic agent.
Examples of suitable erythropoietic agents are erythropoietin
(EPO), such as epoetin alfa (for instance Procrit.RTM.,
Epogen.RTM., and Eprex.RTM.) and epoetin beta (for instance
NeoRecormon.RTM.) and erythropoiesis-stimulating proteins (for
instance Aranesp.RTM.).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be an anti-cancer cytokine,
chemokine, or combination thereof. Examples of suitable cytokines
and growth factors include IFN.gamma., IL-2, IL-4, IL-6, IL-7,
IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a,
IL-28b, IL-29, KGF, IFN.alpha. (e.g., INF.alpha.2b), IFN.beta.,
GM-CSF, CD4OL, Flt3 ligand, stem cell factor, ancestim, and
TNF.alpha.. Suitable chemokines may include Glu-Leu-Arg
(ELR)-negative chemokines such as IP-10, MCP-3, MIG, and
SDF-1.alpha. from the human CXC and C--C chemokine families.
Suitable cytokines include cytokine derivatives, cytokine variants,
cytokine fragments, and cytokine fusion proteins. These and other
methods or uses involving naturally occurring peptide-encoding
nucleic acids herein may alternatively or additionally be performed
by "gene activation" and homologous recombination gene upregulation
techniques, such as are described in U.S. Pat. Nos. 5,968,502,
6,063,630 and 6,187,305 and EP 0505500.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be an agent that modulates, e.g.,
enhances or inhibits, the expression or activity of Fc.alpha. or
Fc.gamma. receptors. Examples of agents suitable for this use
include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6
(IL-6), granulocyte colony-stimulating factor (G-CSF), such as
filgrastim (Neupogen.RTM.) and pegfilgrastim (Neulasta.RTM.), and
granulocyte macrophage-colony stimulating factors (GM-CSF) such as
sargramostim (Leukine.RTM.), interferon-.gamma. (IFN-.gamma.), and
tumor necrosis factor (TNF).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a cell cycle control/apoptosis
regulator (or "regulating agent"). A cell cycle control/apoptosis
regulator may include molecules (i) that target and modulate cell
cycle control/apoptosis regulators such as cdc-25 (such as NSC
663284), (ii) cyclin-dependent kinases that overstimulate the cell
cycle (such as flavopiridol (L868275, HMR1275),
7-hydroxy-staurosporine (UCN-01, KW-2401), and roscovitine
(R-roscovitine, CYC202)), and (iii) telomerase modulators (such as
BIBR1532, SOT-095, GRN163 and compositions described in for
instance U.S. Pat. Nos. 6,440,735 and 6,713,055). Non-limiting
examples of molecules that interfere with apoptotic pathways
include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis-2
ligand (Apo-2L), agents inducing NF-.theta.B blockade leading to
inhibition of IL-6 production, antibodies that activate TRAIL
receptors, IFNs, anti-sense Bcl-2, and As.sub.2O.sub.3 (arsenic
trioxide, Trisenox.RTM.).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a hormonal regulating agent,
such as agents useful for anti-androgen and anti-estrogen therapy.
Examples of such hormonal regulating agents are tamoxifen,
idoxifene, fulvestrant, droloxifene, toremifene, raloxifene,
diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene
(such as flutaminde/eulexin), a progestin (such as such as
hydroxy-progesterone caproate, medroxyprogesterone/provera,
megestrol acepate/megace), an adrenocorticosteroid (such as
hydrocortisone, prednisone), luteinizing hormone-releasing hormone
(and analogs thereof and other LHRH agonists such as buserelin and
goserelin), an aromatase inhibitor (such as anastrazole/arimidex,
aminoglutethimide/cytraden, exemestane), a hormone inhibitor (such
as octreotide/sandostatin) and similar agents.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be an anti-anergic agents (for
instance small molecule compounds, proteins, glycoproteins, or
antibodies that break tolerance to tumor and cancer antigens).
Examples of such compounds are molecules that block the activity of
CTLA-4, such as MDX-010 (Phan et al., PNAS USA 100, 8372
(2003)).
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be a tumor suppressor
gene-containing nucleic acid or vector such as a
replication-deficient adenovirus encoding human recombinant
wild-type p53/SCH58500, etc.; antisense nucleic acids targeted to
oncogenes, mutated, or deregulated genes; or siRNA targeted to
mutated or deregulated genes. Examples of tumor suppressor targets
include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1,
and DCC.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be an anti-cancer nucleic acid,
such as genasense (augmerosen/G3139), LY900003 (ISIS 3521), ISIS
2503, OGX-011 (ISIS 112989), LE-AON/LEraf-AON (liposome
encapsulated c-raf antisense oligonucleotide/ISIS-5132), MG98, and
other antisense nucleic acids that target PKC.alpha., clusterin,
IGFBPs, protein kinase A, cyclin D1, or Bcl-2h.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be an anti-cancer inhibitory RNA
molecule (see for instance Lin et al., Curr Cancer Drug Targets.
1(3), 241-7 (2001), Erratum in: Curr Cancer Drug Targets. 3(3), 237
(2003), Lima et al., Cancer Gene Ther. 11(5), 309-16 (2004), Grzmil
et al., Int J Oncol. 4(1), 97-105 (2004), Collis et al., Int J
Radiat Oncol Biol Phys. 57(2 Suppl), S144 (2003), Yang et al.,
Oncogene. 22(36), 5694-701 (2003) and Zhang et al., Biochem Biophys
Res Commun. 303(4), 1169-78 (2003)).
Compositions and combination administration methods of the present
invention also include the administration of nucleic acid vaccines,
such as naked DNA vaccines encoding such cancer
antigens/tumor-associated antigens (see for instance U.S. Pat. Nos.
5,589,466, 5,593,972, 5,703,057, 5,879,687, 6,235,523, and
6,387,888). In one embodiment, the combination administration
method and/or combination composition comprises an autologous
vaccine composition. In one embodiment, the combination composition
and/or combination administration method comprises a whole cell
vaccine or cytokine-expressing cell (for instance a recombinant
IL-2 expressing fibroblast, recombinant cytokine-expressing
dendritic cell, and the like) (see for instance Kowalczyk et al.,
Acta Biochim Pol. 50(3), 613-24 (2003), Reilly et al., Methods Mol
Med. 69, 233-57 (2002) and Tirapu et al., Curr Gene Ther. 2(1),
79-89 (2002). Another example of such an autologous cell approach
that may be useful in combination methods of the present invention
is the MyVax.RTM. Personalized Immunotherapy method (previously
referred to as GTOP-99) (Genitope Corporation--Redwood City,
Calif., USA).
In one embodiment, the present invention provides combination
compositions and combination administration methods wherein an
anti-CD38 antibody is combined or co-administered with an oncolytic
virus.
Combination compositions and combination administration methods of
the present invention may also involve "whole cell and "adoptive"
immunotherapy methods. For instance, such methods may comprise
infusion or re-infusion of immune system cells (for instance
tumor-infiltrating lymphocytes (TILs), such as CD4.sup.+ and/or
CD8.sup.+ T cells (for instance T cells expanded with
tumor-specific antigens and/or genetic enhancements),
antibody-expressing B cells or other antibody producing/presenting
cells, dendritic cells (e.g., anti-cytokine expressing recombinant
dendritic cells, dendritic cells cultured with a DC-expanding agent
such as GM-CSF and/or Flt3-L, and/or tumor-associated
antigen-loaded dendritic cells), anti-tumor NK cells, so-called
hybrid cells, or combinations thereof. Cell lysates may also be
useful in such methods and compositions. Cellular "vaccines" in
clinical trials that may be useful in such aspects include
Canvaxin.TM., APC-8015 (Dendreon), HSPPC-96 (Antigenics), and
Melacine.RTM. cell lysates. Antigens shed from cancer cells, and
mixtures thereof (see for instance Bystryn et al., Clinical Cancer
Research Vol. 7, 1882-1887, July 2001), optionally admixed with
adjuvants such as alum, may also be components in such methods and
combination compositions.
In one embodiment, an anti-CD38 antibody of the present invention
may be delivered to a patient in combination with the application
of an internal vaccination method. Internal vaccination refers to
induced tumor or cancer cell death, such as drug-induced or
radiation-induced cell death of tumor cells, in a patient, that
typically leads to elicitation of an immune response directed
towards (i) the tumor cells as a whole or (ii) parts of the tumor
cells including (a) secreted proteins, glycoproteins or other
products, (b) membrane-associated proteins or glycoproteins or
other components associated with or inserted in membranes, and/or
(c) intracellular proteins or other intracellular components. An
internal vaccination-induced immune response may be humoral (i.e.
antibody--complement-mediated) or cell-mediated (e.g., the
development and/or increase of endogenous cytotoxic T lymphocytes
that recognize the internally killed tumor cells or parts thereof).
In addition to radiotherapy, non-limiting examples of drugs and
agents that may be used to induce said tumor cell-death and
internal vaccination are conventional chemotherapeutic agents,
cell-cycle inhibitors, anti-angiogenesis drugs, monoclonal
antibodies, apoptosis-inducing agents, and signal transduction
inhibitors.
Examples of other anti-cancer agents, which may be relevant as
therapeutic agents for use in combination with the anti-CD38
antibody of the present invention for treating the disorders as
described above are differentiation inducing agents, retinoic acid
and retinoic acid analogues (such as all trans retinoic acid,
13-cis retinoic acid and similar agents), vitamin D analogues (such
as seocalcitol and similar agents), inhibitors of ErbB3, ErbB4,
IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1,
FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph,
Ret, Ros, Alk, LTK, PTK7 and similar agents.
Examples of other anti-cancer agents, which may be relevant as
therapeutic agents for use in combination with the anti-CD38
antibody of the present invention for treating the disorders as
described above are cathepsin B, modulators of cathepsin D
dehydrogenase activity, glutathione-S-transferase (such as
glutacylcysteine synthetase and lactate dehydrogenase),
estramustine, epirubicin, HSP90 inhibitor like 17-allyl amino
geld-anamycin, antibodies directed against a tumor antigen such as
PSA, CA125, KSA, etc., integrins like integrin .beta.1, inhibitors
of VCAM and similar agents.
Examples of other anti-cancer agents, which may be relevant as
therapeutic agents for use in combination with the anti-CD38
antibodies of the present invention for treating the disorders as
described above are calcineurin-inhibitors (such as valspodar, PSC
833 and other MDR-1 or p-glycoprotein inhibitors), TOR-inhibitors
(such as sirolimus, everolimus and rapamcyin). and inhibitors of
"lymphocyte homing" mechanisms (such as FTY720), and agents with
effects on cell signaling such as adhesion molecule inhibitors (for
instance anti-LFA, etc.).
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38, such as
cancer, in a subject, which method comprises administration of a
therapeutically effective amount of an anti-CD38 antibody of the
present invention and radiotherapy to a subject in need
thereof.
In one embodiment, the present invention provides a method for
treating multiple myeloma, which method comprises administration of
a therapeutically effective amount of an anti-CD38 antibody of the
present invention and radiotherapy to a subject in need
thereof.
Radiotherapy may comprise radiation or associated administration of
radiopharmaceuticals to a patient is provided. The source of
radiation may be either external or internal to the patient being
treated (radiation treatment may, for example, be in the form of
external beam radiation therapy (EBRT), brachytherapy (BT) or
skeletal targeted radiotherapy). Radioactive elements that may be
used in practicing such methods include, e.g., radium, cesium-137,
iridium-192, americium-241, gold-198, cobalt-57, copper-67,
technetium-99, iodide-123, iodide-131, and indium-111.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
to a subject in need thereof combined with autologous peripheral
stem cell or bone marrow transplantation.
In one embodiment, the present invention provides a method for
treating multiple myeloma, which method comprises administration of
a therapeutically effective amount of an anti-CD38 antibody of the
present invention to a subject in need thereof combined with
autologous peripheral stem cell or bone marrow transplantation.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
to a subject in need thereof combined with orthopedic
intervention.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one anti-inflammatory agent to a subject in need
thereof.
In one embodiment such an anti-inflammatory agent may be selected
from a steroidal drug and a NSAID (nonsteroidal anti-inflammatory
drug).
In one embodiment such an anti-inflammatory agent may be selected
from aspirin and other salicylates, Cox-2 inhibitors (such as
rofecoxib and celecoxib), NSAIDs (such as ibuprofen, fenoprofen,
naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal,
nabumetone, etodolac, oxaprozin, and indomethacin), anti-IL6R
antibodies, anti-IL8 antibodies (e.g. antibodes described in
WO2004058797, e.g. 10F8), anti-IL15 antibodies (e.g. antibodies
described in WO03017935 and WO2004076620), anti-IL15R antibodies,
anti-CD4 antibodies (e.g. zanolimumab), anti-CD11a antibodies
(e.g., efalizumab), anti-alpha-4/beta-1 integrin (VLA4) antibodies
(e.g. natalizumab), CTLA4-Ig for the treatment of inflammatory
diseases, prednisolone, prednisone, disease modifying antirheumatic
drugs (DMARDs) such as methotrexate, hydroxychloroquine,
sulfasalazine, pyrimidine synthesis inhibitors (such as
leflunomide), IL-1 receptor blocking agents (such as anakinra),
TNF-.alpha. blocking agents (such as etanercept, infliximab, and
adalimumab) and similar agents.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and at least one immunosuppressive and/or immunomodulatory agent to
a subject in need thereof.
In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from cyclosporine,
azathioprine, mycophenolic acid, mycophenolate mofetil,
corticosteroids such as prednisone, methotrexate, gold salts,
sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,
15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,
tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin,
thymosin-.alpha. and similar agents.
In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from immunosuppressive
antibodies, such as antibodies binding to p75 of the IL-2 receptor,
or antibodies binding to for instance MHC, CD2, CD3, CD4, CD7,
CD28, B7, CD40, CD45, IFN.gamma., TNF-.alpha., IL-4, IL-5, IL-6R,
IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a, or CD58, or antibodies
binding to their ligands.
In one embodiment, such an immunosuppressive and/or
immunomodulatory agent may be selected from soluble IL-15R, IL-10,
B7 molecules (B7-1, B7-2, variants thereof, and fragments thereof),
ICOS, and OX40, an inhibitor of a negative T cell regulator (such
as an antibody against CTLA4) and similar agents.
In one embodiment, the anti-CD38 antibody of the present invention
may be administered in combination with two or more
immunosuppressive and/or immunomodulatory agents, such as in
combination with prednisone and cyclosporine; prednisone,
cyclosporine and azathioprine; or prednisone, cyclosporine and
mycophenolate mofetil.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and an anti-C3b(i) antibody to a subject in need thereof.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
and an anti-CD32b antibody to a subject in need thereof. In one
embodiment of the present invention, the anti-CD32b antibody is
selected from HuMab-016, -020, -022, -024, 026, 028, -034, -038
or-053 all disclosed in WO2009/083009.
In one embodiment, a therapeutic agent for use in combination with
the anti-CD38 antibody of the present invention for treating the
disorders as described above may be selected from histone
deacetylase inhibitors (for instance phenylbutyrate) and/or DNA
repair agents (for instance DNA repair enzymes and related
compositions such as dimericine).
Methods of the present invention for treating a disorder as
described above comprising administration of a therapeutically
effective amount of an anti-CD38 antibody of the present invention
may also comprise anti-cancer directed photodynamic therapy (for
instance anti-cancer laser therapy--which optionally may be
practiced with the use of photosensitizing agent, see, for instance
Zhang et al., J Control Release. 93(2), 141-50 (2003)), anti-cancer
sound-wave and shock-wave therapies (see for instance Kambe et al.,
Hum Cell. 10(1), 87-94 (1997)), and/or anti-cancer nutraceutical
therapy (see for instance Roudebush et al., Vet Clin North Am Small
Anim Pract. 34(1), 249-69, viii (2004) and Rafi, Nutrition. 20(1),
78-82 (2004). Likewise, an anti-CD38 antibody of the present
invention may be used for the preparation of a pharmaceutical
composition for treating a disorder as described above to be
administered with anti-cancer directed photodynamic therapy (for
instance anti-cancer laser therapy--which optionally may be
practiced with the use of photosensitizing agent, anti-cancer
sound-wave and shock-wave therapies, and/or anti-cancer
nutraceutical therapy.
In a further embodiment, the anti-CD38 antibody of the present
invention is administered together with complement.
As described above, a pharmaceutical composition of the present
invention may be administered in combination therapy, i.e.,
combined with one or more agents relevant for the disease or
condition to be treated either as separate pharmaceutical
compositions or with a compound of the present invention
coformulated with one or more additional therapeutic agents as
described above. Such combination therapies may require lower
dosages of the compound of the present invention and/or the
co-administered agents, thus avoiding possible toxicities or
complications associated with the various monotherapies.
In one embodiment, the present invention provides a method for
treating a disorder involving cells expressing CD38 in a subject,
which method comprises administation of a therapeutically effective
amount of an anti-CD38 antibody of the present invention and at
least one immunosuppressive and/or immunomodulatory agent to a
subject in need thereof
Diagnostic Uses
The anti-CD38 antibodies of the invention may also be used for
diagnostic purposes. Thus, in a further aspect, the invention
relates to a diagnostic composition comprising an anti-CD38
antibody as defined herein.
In one embodiment, the anti-CD38 antibodies of the present
invention may be used in vivo or in vitro for diagnosing diseases
wherein activated cells expressing CD38 play an active role in the
pathogenesis, by detecting levels of CD38, or levels of cells which
contain CD38 on their membrane surface. This may be achieved, for
example, by contacting a sample to be tested, optionally along with
a control sample, with the anti-CD38 antibody under conditions that
allow for formation of a complex between the antibody and CD38.
Complex formation is then detected (e.g., using an ELISA). When
using a control sample along with the test sample, complex is
detected in both samples and any statistically significant
difference in the formation of complexes between the samples is
indicative of the presence of CD38 in the test sample.
Thus, in a further aspect, the invention relates to a method for
detecting the presence of CD38 antigen, or a cell expressing CD38,
in a sample comprising: contacting the sample with an anti-CD38
antibody of the invention or a bispecific molecule of the
invention, under conditions that allow for formation of a complex
between the antibody and CD38; and analyzing whether a complex has
been formed.
In one embodiment, the method is performed in vitro.
More specifically, the present invention provides methods for the
identification of, and diagnosis of invasive cells and tissues, and
other cells targeted by anti-CD38 antibodies of the present
invention, and for the monitoring of the progress of therapeutic
treatments, status after treatment, risk of developing cancer,
cancer progression, and the like.
In one example of such a diagnostic assay, the present invention
provides a method of diagnosing the level of invasive cells in a
tissue comprising forming an immunocomplex between an anti-CD38
antibody and potential CD38-containing tissues, and detecting
formation of the immunocomplex, wherein the formation of the
immunocomplex correlates with the presence of invasive cells in the
tissue. The contacting may be performed in vivo, using labeled
isolated antibodies and standard imaging techniques, or may be
performed in vitro on tissue samples.
Examples of conventional immunoassays provided by the present
invention include, without limitation, an ELISA, an RIA, FACS
assays, plasmon resonance assays, chromatographic assays, tissue
immunohistochemistry, Western blot, and/or immunoprecipitation
using an anti-CD38 antibody. Suitable labels for the anti-CD38
antibody and/or secondary antibodies used in such techniques
include, without limitation, various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, and radioactive
materials.
The anti-CD38 antibodies are particularly useful in the in vivo
imaging of tumors. In vivo imaging of tumors associated with CD38
may be performed by any suitable technique. For example,
.sup.99Tc-labeling or labeling with another gamma-ray emitting
isotope may be used to label anti-CD38 antibodies in tumors or
secondary labeled (e.g., FITC labeled) anti-CD38 antibody:CD38
complexes from tumors and imaged with a gamma scintillation camera
(e.g., an Elscint Apex 409ECT device), typically using low-energy,
high resolution collimator or a low-energy all-purpose collimator.
Stained tissues may then be assessed for radioactivity counting as
an indicator of the amount of CD38-associated peptides in the
tumor. The images obtained by the use of such techniques may be
used to assess biodistribution of CD38 in a patient, mammal, or
tissue, for example in the context of using CD38 or CD38-fragments
as a biomarker for the presence of invasive cancer cells.
Variations on this technique may include the use of magnetic
resonance imaging (MRI) to improve imaging over gamma camera
techniques. Similar immunoscintigraphy methods and principles are
described in, e.g., Srivastava (ed.), Radiolabeled Monoclonal
Antibodies For Imaging And Therapy (Plenum Press 1988), Chase,
"Medical Applications of Radioisotopes," in Remington's
Pharmaceutical Sciences, 18th Edition, Gennaro et al., (eds.), pp.
624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of
Monoclonal Antibodies," in Biotechnology And Pharmacy 227-49,
Pezzuto et al., (eds.) (Chapman & Hall 1993).
In a further aspect, the invention relates to a kit for detecting
the presence of CD38 antigen, or a cell expressing CD38, in a
sample comprising an anti-CD38 antibody of the invention or a
bispecific molecule of the invention; and instructions for use of
the kit.
In one embodiment, the present invention provides a kit for
diagnosis of cancer comprising a container comprising an anti-CD38
antibody, and one or more reagents for detecting binding of the
anti-CD38 antibody to a CD38 peptide. Reagents may include, for
example, fluorescent tags, enzymatic tags, or other detectable
tags. The reagents may also include secondary or tertiary
antibodies or reagents for enzymatic reactions, wherein the
enzymatic reactions produce a product that may be visualized. In
one embodiment, the present invention provides a diagnostic kit
comprising one or more anti-CD38 antibodies of the present
invention in labeled or unlabeled form in suitable container(s),
reagents for the incubations for an indirect assay, and substrates
or derivatizing agents for detection in such an assay, depending on
the nature of the label. Control reagent(s) may also be
included.
In a further aspect, the invention relates to an anti-idiotypic
antibody which binds to an anti-CD38 antibody of the invention as
described herein.
An anti-idiotypic (Id) antibody is an antibody which recognizes
unique determinants generally associated with the antigen-binding
site of an antibody. An Id antibody may be prepared by immunizing
an animal of the same species and genetic type as the source of an
anti-CD38 mAb with the mAb to which an anti-Id is being prepared.
The immunized animal typically can recognize and respond to the
idiotypic determinants of the immunizing antibody by producing an
antibody to these idiotypic determinants (the anti-Id antibody).
Such antibodies are described in for instance U.S. Pat. No.
4,699,880.
An anti-Id antibody may also be used as an "immunogen" to induce an
immune response in yet another animal, producing a so-called
anti-anti-Id antibody. An anti-anti-Id may be epitopically
identical to the original mAb, which induced the anti-Id. Thus, by
using antibodies to the idiotypic determinants of a mAb, it is
possible to identify other clones expressing antibodies of
identical specificity. Anti-Id antibodies may be varied (thereby
producing anti-Id antibody variants) and/or derivatized by any
suitable technique, such as those described elsewhere herein with
respect to anti-CD38 antibodies of the present invention.
The present invention is further illustrated by the following
examples which should not be construed as further limiting.
EXAMPLES
Example 1
Generation of Antibodies
HCo12 mice were immunized every fortnight with 20 .mu.g purified
HA-CD38 alternating with NIH-3T3-CD38 transfected cells. The first
immunization was performed with 5.times.10.sup.6 cells in 100 .mu.l
PBS, mixed with 100 .mu.l CFA, i.p., the second and following
immunizations with HA-CD38 s.c., in the presence of 100 .mu.l PBS,
mixed with 100 .mu.l IFA. The following immunizations with
transfected cells were performed in the presence of 200 .mu.l PBS.
After titer development, mice were boosted with 20 .mu.g HA-CD38 in
PBS, i.v.
Spleens were harversted from these mice, splenocytes were isolated
and fused with PEG to a mouse myeloma cell line based upon standard
protocols. The resulting hybridomas were then screened for human
antibody production by ELISA and for CD38 specificity using human
CD38-transfected NS/0 cells by FACS analysis and recombinant
HA-CD38 protein binding by ELISA.
Sequence Analysis of the anti-CD38 HuMab Variable Domanins and
Cloning in Expression Vectors
Total RNA of the anti-CD38 HuMabs was prepared from
5.times.10.sup.6 hybridoma cells and 5'-RACE-Complementary DNA
(cDNA) was prepared from 100 ng total RNA, using the SMART RACE
cDNA Amplification kit (Clontech), according to the manufacturer's
instructions.
VH and VL coding regions were amplified by PCR and cloned into the
pCR-Blunt II-TOPO vector (Invitrogen) using the Zero Blunt PCR
cloning kit (Invitrogen). For each HuMab, 16 VL clones and 8 VH
clones were sequenced.
The VL and VH encoding regions were cloned into the pKappa and pG1f
vectors.
CDR regions are indicated according to IMGT.
(http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=humanIg)
The following IgG1,.kappa. human monoclonal antibodies were
identified:
TABLE-US-00005 VH VL 025 SEQ ID NO: 2 SEQ ID NO: 27 026 SEQ ID NO:
7 SEQ ID NO: 27 028 SEQ ID NO: 12 SEQ ID NO: 37 049 SEQ ID NO: 17
SEQ ID NO: 42 056 SEQ ID NO: 22 SEQ ID NO: 47
Example 2
Electrospray Ionisation-Quadrupole-Time of Flight Mass Spectrometry
of Anti-CD38 Antibodies
Intact molecular weight data for anti-CD38 antibodies 025, 057
(same amino acid sequence as antibody 026), 028, 049 and 056 were
obtained using nanospray Electrospray-MS on a Q-TOF mass
spectrometer. Aliquots of each antibody sample were desalted
offline using C.sub.4 micro-tap cartridge and eluted in
propanol/trifluoroacetic acid solvent. The instrument was
calibrated using glu-fibrinopeptide fragment ions in MS/MS mode.
MassLynx 4.0 software was used to de-convolute the multiply-charged
data obtained.
Information on the molecular weight of light and heavy chain
components of these antibodies was obtained following reduction
using dithiothreitol and analysis as described above.
TABLE-US-00006 TABLE 1 Mass of CD38 antibodies (in Dalton) Anti-
intact MW Light Heavy Chain body K0 K1 K2 Chain K0 K1 -025 144742.8
144874.1 144999.8 23357.8 49017.4 49145.8 -057 144828.2 144946.4
145071.2 23357.8 49047.4 49175.8 -028 144818.3 144946.4 145074.7
23357.8 49054.5 49182.8 -049 144864.0 144990.6 145117.8 23384.8
49049.3 49177.9 -056 145100.7 145222.7 23384.8 49099.5 49226.4
Example 3
Cross-Block Studies using FACS
CHO-CD38 cells were incubated with an excess of unlabelled
CD38-specific antibodies (4.degree. C., 15 min). Then, cells were
incubated with FITC-labeled 005 antibody (concentration
approximates EC.sub.90, 4.degree. C., 45 min) (005 is disclosed in
WO2006099875). After washing the cells twice with PBS-BSA,
fluorescence was measured by flow cytometry. 005-FITC labeled
antibody binding was blocked by excess unlabelled antibodies 025,
026, 028, 049 and 056, indicating that these antibodies have
overlapping epitopes. Binding of 005-FITC was not blocked by excess
unlabelled 003 (disclosed in WO2006099875) providing evidence for
binding to a different epitope.
Cross-Blocking Studies using ELISA
Soluble human CD38 was coated on the surface of an ELISA plate.
Coated CD38 was incubated with an excess of unlabelled CD38
specific antibodies for about 15 minutes and subsequently
biotinylated CD38-specific antibodies were added (concentration
approximates EC.sub.90, RT, 1 hour). After washing three times with
PBS/Tween, horseradish peroxidase (HRP)-conjugated streptavidine
was added and the mixture was incubated for 1 hour at RT. The
complex was detected by addition of an ABTS-solution and the HRP
mediated substrate conversion was measured using an ELISA reader at
OD 405 nm.
Cross-Blocking Studies using Sandwich-ELISA
Anti-CD38 antibodies were coated on the surface of an ELISA plate.
Plate-bound antibodies were incubated with biotinylated soluble
CD38 in the presence of an excess of anti-CD38 antibodies in fluid
phase. After washing with PBS/Tween, bound biotinylated CD38 was
detected with HRP-conjugated streptavidine for 1 hr at RT. This
complex was detected by addition of an ABTS-solution (after washing
with PBS/Tween) and the HRP mediated substrate conversion was
measured using an ELISA reader at OD 405 nm.
Example 4
Epitope Mapping
Construction of HA-CD38 and His-CD38 Expression Vectors
The encoding sequences for the extracellular domain of human CD38
(identical to amino acids 45-300 from Genbank entry AAA68482) were
amplified from plasmid pCIpuroCD38 (obtained from Prof. M. Glennie,
Tenovus Research Laboratory, Southampton General Hospital,
Southampton, UK) using PCR, introducing, restriction sites, an
ideal Kozak sequence (GCCGCCACC), and sequences endcoding a signal
peptide and a N-terminal HA tag (ypydvpdya). The construct was
cloned in the mammalian expression vector pEE13.4 (Lonza
Biologics). This construct was named pEE13.4HACD38.
A similar construct was made synthetically and fully codon
optimized (GeneArt, Regensburg, Germany), replacing the HA tag
encoding part by a His tag (HHHHHH) encoding part. The construct
was cloned in pEE13.4 and named pEE13.4HisCD38.
Site Directed Mutagenesis
Several mutations were introduced in the putative antibody binding
site on the CD38 molecule.
DNA substitutions leading to T237A, Q272R or S274F amino acid
substitutions were generated using the QuickChange II XL
Site-directed Mutagenesis Kit (Stratagene, Amsterdam, The
Netherlands) in the pEE13.4HACD38 vector. Similarly a D202G
encoding substitution was introduced in the pEE13.4HisCD38
vector.
Transient Expression in HEK-293F cells
Freestyle.TM. 293-F (a HEK-293 subclone adapted to suspension
growth and chemically defined Freestyle medium, (HEK-293F)) cells
were obtained from Invitrogen and transfected with pEE13.4HACD38,
pEE13.4HisCD38 or the four constructs carrying the mutations,
according to the manufacturer's protocol using 293fectin
(Invitrogen). Cell culture supernatants of transfected cells were
used in ELISA for anti-CD38 binding studies.
Anti-CD38 Antibody Binding
Mutations T237A, Q272R, and S274F: ELISA plates (Greiner, #655092)
were coated O/N at 4.degree. C. with 1 .mu.g anti-HA antibody
(Sigma, #H-9658) and subsequently blocked with 2% chicken serum.
Culture supernatants of transfected HEK293F cells were diluted,
applied to the ELISA plates and incubated for 1 hr at RT. After
washing, serial dilutions of anti-CD38 antibodies were added and
incubated for 1 hr at RT. Bound antibodies were detected with
HRP-conjugated goat-anti-human IgG antibodies. The assay was
developed with ABTS (Roche, #1112597) and the absorbance was
measured at 405 nm using a spectrophotometer.
Mutation D202G: ELISA plates (Greiner, #655092) were coated O/N at
4.degree. C. with 1 .mu.g penta-His (Qiagen #34660) and
subsequently blocked with 2% PBS/BSA. Culture supernatants of
transfected HEK293F cells were diluted, applied to the ELISA plates
and incubated for 2 hr at RT. After washing, serial dilutions of
anti-CD38 antibodies were added and incubated for 1 hr at RT. Bound
antibodies were detected with HRP-conjugated goat-anti-human IgG
antibodies. The assay was developed with ABTS (Roche, #1112597) and
the absorbance was measured at 405 nm using a
spectrophotometer.
This study revealed that binding of 025, 026, 028 and 049 was not
sensitive to mutations T237A, Q272R, S274F, and A199T, but was
seriously affected by D202G (025, 028, 049) (FIG. 2).
Example 5
Binding of Anti-CD38 Antibodies to CD38-Transfected CHO (CHO-CD38)
Cells and to Daudi-luc Cells
After harvesting and counting, Daudi-luc cells, CHO cells
transfected with CD38 and control CHO cells, were resuspended in
PBS (1.times.10.sup.6 cells/mL). Cells were transferred to 96-well
V-bottom plates (100 .mu.L/well) and washed twice in PBS-BSA (PBS
supplemented with 0.1% BSA and 0.02% Na-azide). 50 .mu.L antibody
in PBS-BSA was added in three-fold dilutions ranging from 0.3 to 30
.mu.g/mL (4.degree. C., 30 min). After washing three times in
PBS-BSA, 50 .mu.L (1:400 dilution) of rabbit anti-human IgG-FITC in
PBS-BSA was added (4.degree. C. in the dark, 30 minutes). Cells
were washed three times and specific binding of CD38-antibodies to
CHO-CD38 and Daudi-luc cells was detected by flow cytometry.
FIG. 3 shows that 025, 026, 028, 049, and 056 bind to CHO-CD38
cells and to Daudi-luc cells.
Example 6
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
The ability of anti-CD38 antibodies to perform ADCC of Daudi-luc
cells was determined as explained below. As effector cells,
peripheral blood mononuclear cells from healthy volunteers (UMC
Utrecht, The Netherlands) were used.
Daudi-luc cells were collected (5x10.sup.6 cells) in RPMI.sup.++
(RPMI 1640 culture medium supplemented with 10% cosmic calf serum
(HyClone, Logan, Utah, USA)), to which 100 .mu.Ci .sup.51Cr
(Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The
Netherlands) was added, and the mixture was incubated in a
37.degree. C. water bath for 1 hr. After washing of the cells
(twice in PBS, 1500 rpm, 5 min), the cells were resuspended in
RPMI.sup.++ and counted by trypan blue exclusion. Cells were
brought at concentration of 1.times.10.sup.5 cells/mL.
Preparation of Effector Cells
Fresh peripheral blood mononuclear cells (healthy volunteers, UMC
Utrecht, Utrecht, The Netherlands) were isolated from 40 ml of
heparin blood by Ficoll (Bio Whittaker; lymphocyte separation
medium, cat 17-829E) according to the manufacturer's instructions.
After resuspension of cells in RPMI.sup.++, cells were counted by
trypan blue exclusion and brought at concentration of
1.times.10.sup.7 cells/ml.
ADCC Set Up
50 .mu.l of .sup.51Cr-labeled targets cells were pipetted into
96-well plates, and 50 .mu.l of antibody was added, diluted in
RPMI++(final concentrations 10, 1, 0.1, 0.01 .mu.g/ml). Cells were
incubated (RT, 15 min), and 50 .mu.l effector cells were added,
resulting in an effector to target ratio of 100:1 (for
determination of maximal lysis, 100 .mu.l 5% Triton-X100 was added
instead of effector cells; for determination of spontaneous lysis,
50 pL target cells and 100 .mu.L RPMI++ were used). Cells were spun
down (500 rpm, 5 min), and incubated (37.degree. C., 5% CO.sub.2, 4
hr). After spinning down cells (1500 rpm, 5 min), 100 .mu.L of
supernatant was harvested into micronic tubes, and counted in gamma
counter. The percentage specific lysis was calculated as follows:
(cpm sample-cpm target cells only)/(cpm maximal lysis-cpm target
cells only) wherein cpm is counts per minute.
025, 026, 028, 049, and 056 induced ADCC mediated lysis in Daudi
cells (FIG. 4).
Example 7
Complement-Dependent Cytotoxicity (CDC)
After harvesting and counting of Daudi-luc cells, the viability of
the cells should be .gtoreq.90%. After washing (PBS), cells are
resuspended at 2.0.times.10.sup.6 cells/ml in RPMI-B (RPMI
supplemented with 1% BSA). Thereafter, cells are put in 96-well
round-bottom plates at 1.times.10.sup.5 cells/well (50 .mu.L/well).
Then, 50 .mu.L antibodies is added to the wells (final
concentration range between 0-100 .mu.g/ml (three-fold dilutions in
RPMI-B)). After incubation (RT, 15 min), 11 .mu.L of pooled human
serum (pool of 18 healthy donors) was added to each well
(37.degree. C., 45 min). Wells were resuspended once and 120 .mu.L
was transferred to FACS tubes (Greiner). Then, 10 .mu.L propidium
iodide (PI; Sigma-Aldrich Chemie B.V.) was added (10 .mu.g/ml
solution) to this suspension. Lysis was detected by flow cytometry
(FACScalibur.TM., Becton Dickinson, San Diego, Calif., USA) by
measurement of the percentage of dead cells (corresponds to
PI-positive cells).
FIG. 5 presents CDC-mediated CHO-CD38 cell lysis caused by
anti-CD38 antibodies 025, 026, 028, 049 and 056. These anti-CD38
antibodies failed to induce CDC of Daudi-luc cells.
Example 8
Enzymatic Activity
The effects of anti-CD38 antibodies on the enzymatic activities of
CD38 were determined. CD38 is known to catalyze several different
enzymatic reactions, including a cyclase reaction converting NAD
into cyclic ADP ribose (cADPR), a hydrolase reaction converting NAD
or cADPR into ADP ribose, and a base-exchange reaction in which
nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is
produced.
Cyclase Activity
NGD Assay
The ability of anti-CD38 antibodies to interfere with the cyclase
activity of CD38 using NGD as a substrate was measured in an assay
essentially as described in Graeff et al., J. Biol. Chem. 269,
30260-30267 (1994):
Briefly, substrate NGD.sup.+ (80 .mu.M) was incubated with CD38
(0.6 .mu.g/ml His-tagged extracellular domain of human CD38, see
Example 3 of W02006099875 regarding purification of His-CD38 in a
buffer containing 20 mM Tris-HCl, pH 7.0). The production of cGDPR
can be monitored spectrophotometrically at the emission wavelength
of 410 nm (excitation at 300 nm). In this example an excitation
filter of 340.+-.60 nm and an emission filter of 430.+-.8 nm were
used.
To test the effect of 025, 026, 028, 049 and 056 on the enzymatic
activity of CD38, recombinant His-CD38 protein was pre-incubated
for 15 minutes at room temperature with 3 .mu.g/ml of the
antibodies before adding the substrate NGD.sup.+. The production of
cyclic GDP-ribose (cGDPR) was recorded after 90 minutes.
FIG. 6A shows that antibodies 025, 026, 028, 049 and 056 have a
pronounced inhibitory effect on the production of cGDPR. After 90
minutes, 3 .mu.g/ml of antibody (025, 026, 028, 049 or 056)
resulted in a 53-66% reduced production of cGDPR. In a time course
experiment it was shown that the rate of cGDPR production was
reduced in samples treated with the CD38-specific antibody mAb 028
compared to the cGDPR production in the presence of HuMab-KLH or in
the untreated CD38 control (FIG. 6B). FIG. 6C shows a dose response
curve (0.01-30 .mu.g/ml) for antibody 028. In this experiment a
maximum reduction of cGDPR production of 41% is observed.
To test the effect of 028 on the enzymatic activity of cellular
expressed CD38, CHO-CDC38 cells were pre-incubated for 30 minutes
at room temperature with a serial dilution of 028 (0.01-30
.mu.g/ml) before adding the substrate NGD.sup.+. The production of
cyclic GDP-ribose (cGDPR) was recorded after 90 min. As shown in
FIG. 6D antibody 028 inhibits the production of cGDPR in a
concentration dependent fashion.
Reverse Cyclase Reaction
The effect of mAb 028 on cADPR production from NAD by CD38 was
determined by the reverse cyclase reaction. This assay is based on
the reversibility of the reaction catalyzed by CD38. In the
presence of high concentrations of nicotinamide and cADPR, the
ADP-ribosyl cyclases can produce NAD. Antibodies were diluted to 10
.mu.g/ml in 20 mM Tris-HCl, 0.01% (v/v) BSA, pH 7.2 (Tris/BSA).
Human recombinant CD38 was diluted to 2 .mu.g/ml with Tris/BSA. The
antibodies were preincubated for 10 minutes with CD38 by mixing
equal volumes (50 .mu.L) of the diluted antibodies with the diluted
CD38. The preincubation was done at room temperature. The reaction
was initiated by transferring 25 .mu.L of CD38/antibody mixture to
25 .mu.L of a solution containing 1 mM cADPR and 10 mM
nicotinamide. The reaction was allowed to proceed at room
temperature for 1 to 20 minutes and was stopped at the appropriate
time by filtering the entire sample through a Millipore
MultiScreen-IP Filter 96-well plate to remove protein. The
resulting NAD produced was measured by the method of Graeff and Lee
(1). Controls containing nicotinamide without cADPR were run to
estimate the amount NAD contaminating the reagents. In these
experiments there was undetectable contaminating NAD.
Table 2 shows that 1 .mu.g/ml mAb-028 reduced cADPR production from
NAD by 67%. mAb-KLH had no effect on cADPR production from NAD.
TABLE-US-00007 TABLE 2 The effect of antibody 028 on cADPR
production from NAD pmol cADPR/ Condition min CD38 control 4.3
mAb-KLH 4.3 mAb-028 1.4
8-amino-NAD (8NH2-NAD) Assay
As cADPR production only accounts for approximately 1% of the
product generated from NAD by CD38 (ADPR accounts for the rest),
ribosyl cyclase activity was also assessed using 8-amino-NAD
(8NH2-NAD) as a substrate. Unlike NAD, a considerably larger amount
(approximately 8%) of the 8NH2-NAD substrate is cyclized to
8-amino-cADPR (8NH2-cADPR) and is detectable by HPLC analysis.
Briefly, antibodies were diluted to 10 .mu.g/mL in 20mM Tris-HCl,
0.01% (v/v) BSA, pH 7.2 (Tris/BSA). Human recombinant CD38 was
diluted to 2 .mu.g/ml with Tris/BSA. The antibodies were
preincubated for 10 minutes with CD38 by mixing equal volumes (50
.mu.L) of the diluted antibodies with the diluted CD38. The
preincubation was done at room temperature. The reaction was
initiated by transferring 25 pL of CD38/antibody mixture to 75
.mu.L of 0.5 mM 8NH2-NAD. The reaction was allowed to proceed at
room temperature for 10 minutes and was stopped at the appropriate
time by filtering the entire sample through a Millipore
MultiScreen-IP Filter 96-well plate to remove protein. The reaction
products (8NH2-cADPR and 8NH2-ADPR) were analyzed by reverse phase
HPLC as follows. The column was a 0.46.times.15 cm LC18-T reverse
phase column from Supelco. Solvent A consisted of 20 mM KH2PO4, 5
mM tetrabutylammonium phosphate, pH 6 and solvent B consisted of
50% A and 50% methanol. The flow rate was 1 mL/min and the initial
composition of solvents was 15% B. Separation of substrates and
products was accomplished using the following gradient: 0 to 3.5
minutes (15% B), 3.5 to 5.5 minutes (15 to 32.5% B), 5.5 to 9
minutes (32.5 to 40% B), 9 to 11.5 minutes (40 to 50% B) and 16 to
18 minutes (50 to 15% B) gradient was used to elute the substrates
and products. Samples were prepared by adding 400 .mu.L of solvent
A to 100 .mu.L of filtered sample. The entire sample was injected.
The flow rate and buffer composition were controlled by Beckman 125
HPLC pumps and System Gold software and peaks were detected with a
Beckman 166 UV detector. The areas of the 8NH2-NAD, 8NH2-cADPR and
8NH2-ADPR peaks were used to calculate the amount of 8NH2-cADPR
produced in the assay. The HPLC system is based on a system
described by Schweitzer et al. (2).
FIG. 7A shows that mAb-028 inhibits 8NH2-cADPR by 78%. mAb-028
inhibits 8NH2-cADPR production in a concentration dependent manner
(FIG. 7B)
Thus mAb-028 inhibits the ADP-ribosyl cyclase reaction of CD38 as
assayed by three different methods.
Hydrolase Activity
Hydrolase Activity Analysis by HPLC
The hydrolase activity was measured by determining the amount of
ADPR produced from cADPR or NAD by HPLC. Antibodies were diluted to
10 .mu.g/mL or titrated in 20 mM Tris-HCl, 0.01% (v/v) BSA, pH 7.2
(Tris/BSA). Human recombinant CD38 was diluted to 2 .mu.g/mL with
Tris/BSA. The antibodies were preincubated for 10 minutes with CD38
by mixing equal volumes (50 .mu.L) of the diluted antibodies with
the diluted CD38. The preincubation was done at room temperature.
For the HPLC-based method the cADPR hydrolase reaction was
initiated by transferring 40 .mu.L of CD38/antibody mixture to 10
.mu.L of 4.3 mM cADPR and the NADase reaction was initiated by
transferring 40 .mu.L of CD38/antibody mixture to 10 .mu.L 1 mM
NAD. The reaction was allowed to proceed at room temperature and
was stopped at the appropriate time by adding 25 .mu.L of 1 M HCl.
Protein was removed by filtering the entire sample through a
Millipore MultiScreen-IP Filter 96-well plate. Each filtrate was
neutralized by adding 15 .mu.L of 2M Tris-base and kept on ice
until analyzed by HPLC. The anaylsis of hydrolase activity is based
on the HPLC assay developed by Lee and Aarhus (3). The samples were
analyzed on a 0.5.times.5cm column of AG MP-1 (trifluoroacetate
form) eluted at 3 mL/min with a 0 to 150 mM concave upward gradient
of trifluoroacetic acid (TFA) over 10 minutes. The flow rate and
buffer composition were controlled by Beckman 125 HPLC pumps and
System Gold software and peaks were detected with a Beckman 166 UV
detector. The areas of NAD, cADPR and ADPR were used to calculate
the amount of ADPR produced in the assay.
At concentrations of 10 .mu.g/mL mAb-028, but not mAb-KLH,
stimulated the cADPR hydrolase activity by 62% and the NAD
hydrolase activity by 37% compared to the CD38 control (FIG. 8A).
FIG. 8B shows that mAb-028 stimulated cADPR hydrolysis in a
dose-dependent manner. At concentrations of 30 .mu.g/mL, mAb-028
stimulated hydrolase activity by 78%.
Hydrolase Activity Analysis by Thin Layer Chromatography (TLC)
The hydrolase activity was measured by measuring the amount of
.sup.32P-ADPR produced from .sup.32P-cADPR by thin layer
chromatography (4). The .sup.32P-based cADPR hydrolase reaction was
initiated by adding 20 .mu.L of CD38/antibody mixture (as above) to
5 .mu.L of a mixture containing 0.5 mM cADPR and approximately 0.1
.mu.Ci of .sup.32P-cADPR. The reaction was allowed to proceed at
room temperature and at the appropriate times, 5 .mu.L of the
reaction was added to 5 .mu.L of 150 mM TFA to stop the reaction.
The reaction was analyzed by PEI-cellulose thin layer
chromatography (TLC). One (1) .mu.L of each stopped reaction sample
was spotted on the origin of a PEI--cellulose TLC plate
(10.times.20 cm). The plates were developed with 0.2 M NaCl in 30%
(v/v) ethanol. The plates were dried and exposed to phosphoimage
screens. The screens were analyzed on a Packard Cyclone
Phosphorimager to determine the amount of .sup.32P-ADPR
produced.
FIG. 8C shows that mAb-028 stimulated .sup.32P-cADPR hydrolysis in
a dose-dependent manner. These results were similar to the results
of the cADPR hydrolase activity measured by HPLC (see FIG. 8B).
Base-Exchange Activity
The effect of CD38 antibodies on nicotinic acid adenine
dinucleotide 2'-phosphate (NAADP) synthesis by the base-exchange
activity of CD38 was assessed. Antibodies (mAb-KLH and 028) were
diluted to 40 .mu.g/mL in 20 mM Hepes, pH 7.3, 0.01% (v/v) BSA
(Hepes/BSA). Human recombinant CD38 was diluted to 2 .mu.g/mL with
Hepes/BSA. The antibodies were preincubated for 10 minutes with
CD38 by mixing equal volumes (90 .mu.L) of the diluted antibodies
with the diluted CD38. The preincubation was performed at room
temperature. The base-exchange reaction was initiated by
transferring 50 .mu.L of CD38/antibody mixture to 50 .mu.L of a
reaction mixture containing 200 mM sodium acetate, pH 4.0, 25 mM
nicotinic acid and 2 mM nicotinamide adenine dinucleotide
2'-phosphate (NADP). The reaction was allowed to proceed at room
temperature for 30 minutes and was stopped by filtering the entire
sample through a Millipore MultiScreen-IP Filter 96-well plate to
remove protein. The reaction products in the filtrates were
determined by anion-exchange HPLC on a 0.5.times.5cm column of AG
MP-1 (trifluoroacetate form) eluted at 1 mL/min with a 0 to 150 mM
concave upward gradient of trifluoroacetic acid (TFA) over 30
minutes (5). The filtrates (50 .mu.L) were neutralized by adding 5
.mu.L of 2 M Tris-base just prior to injection. The flow rate and
buffer composition were controlled by Beckman 125 HPLC pumps and
System Gold software and peaks were detected with a Beckman 166 UV
detector. The areas of the NADP, NAADP (base-exchange product) and
adenosine diphospho-ribose 2'-phosphate (ADPR-P, hydrolytic
product) peaks were used to calculate the rates of NAADP synthesis
and NADP hydrolysis.
FIG. 9 shows that mAb-028 inhibits the ability of CD38 to catalyze
the formation of NAADP. The inhibition of NAADP production by
mAb-028 is concentration dependent (FIG. 9B) with an IC50 of 0.14
.mu.g/mL.
LIST OF REFERENCES
1. Graeff, R., and H. C. Lee. 2002. A novel cycling assay for
cellular cADP-ribose with nanomolar sensitivity. Biochem J
361:379-384. 2. Schweitzer, K., G. W. Mayr, and A. H. Guse. 2001.
Assay for ADP-ribosyl cyclase by reverse-phase high-performance
liquid chromatography. Anal Biochem 299:218-226. 3. Lee, H. C., and
R. Aarhus. 1993. Wide distribution of an enzyme that catalyzes the
hydrolysis of cyclic ADP-ribose. Biochim Biophys Acta 1164:68-74.
4. White, T. A., S. Johnson, T. F. Walseth, H. C. Lee, R. M.
Graeff, C. B. Munshi, Y. S. Prakash, G. C. Sieck, and M. S. Kannan.
2000. Subcellular localization of cyclic ADP-ribosyl cyclase and
cyclic ADP-ribose hydrolase activities in porcine airway smooth
muscle. Biochim Biophys Acta 1498:64-71. 5. Aarhus, R., R. M.
Graeff, D. M. Dickey, T. F. Walseth, and H. C. Lee. 1995.
ADP-ribosyl cyclase and CD38 catalyze the synthesis of a
calcium-mobilizing metabolite from NADP. J Biol Chem
270:30327-30333.
SEQUENCE LISTINGS
1
551363DNAhomo sapiens 1caggtccaac tggtgcagtc tggggctgag gtgaagaagc
ctgggtcctc ggtgaaggtc 60tcctgcaagg cttttggagg caccttcagc agctacgcta
tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaagg
atcatccgtt tccttggtat agcaaactac 180gcacagaagt tccagggcag
agtcacgctt atcgcggaca aatccacgaa cacagcctac 240atggagctga
gcagcctgag atctgaggac acggccgttt attactgtgc gggggaacct
300ggggagcggg accccgatgc tgttgatatc tggggccaag ggacaatggt
caccgtctct 360tca 3632121PRThomo sapiens 2Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Phe Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile
Ile Arg Phe Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly
Arg Val Thr Leu Ile Ala Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Gly Glu Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile Trp
Gly 100 105 110Gln Gly Thr Met Val Thr Val Ser Ser 115 12039PRThomo
sapiens 3Gly Gly Thr Ser Phe Ser Ser Tyr Ala1 548PRThomo sapiens
4Ile Ile Arg Phe Leu Gly Ile Ala1 5514PRThomo sapiens 5Ala Gly Glu
Pro Gly Glu Arg Asp Pro Asp Ala Val Asp Ile1 5 106364DNAhomo
sapiens 6caggtccaac tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc
ggtgaaggtc 60tcctgcaagg cttttggagg caccttcagc agctatgcta tcagctgggt
acgacaggcc 120cctggacaag ggcttgagtg gatgggaagg atcatccgtt
tccttggtaa agcaaatcac 180gcacagaagt tccagggcag agtcacgctt
accgcggaca aatccacgaa cacagcctac 240atggagctga gcagcctgag
atctgaggac acggccgttt attactgtgc gggggaacct 300ggggatcggg
accccgatgc tgttgatatc tggggccaag ggacaatggt caccgtctct 360tcag
3647121PRThomo sapiens 7Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Phe Gly
Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Arg Phe Leu Gly
Lys Ala Asn His Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr
Ala Asp Lys Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly Glu Pro
Gly Asp Arg Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110Gln Gly
Thr Met Val Thr Val Ser Ser 115 12088PRThomo sapiens 8Gly Gly Thr
Phe Ser Ser Tyr Ala1 598PRThomo sapiens 9Ile Ile Arg Phe Leu Gly
Lys Ala1 51014PRThomo sapiens 10Ala Gly Glu Pro Gly Asp Arg Asp Pro
Asp Ala Val Asp Ile1 5 1011364DNAhomo sapiens 11caggtccaac
tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg
cttttggagg caccttcagc agttatgcta ttagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaagg atcatccgtt tccttggtaa
aacaaatcac 180gcacagaagt tccagggcag agtcacactt accgcggaca
aatccacgaa cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgttt attactgtgc gggggaacct 300ggggatcggg accccgatgc
tgttgatatc tggggccaag ggacaatggt caccgtctct 360tcag 36412121PRThomo
sapiens 12Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Phe Gly Gly Thr Phe
Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Arg Phe Leu Gly Lys Thr Asn
His Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Lys
Ser Thr Asn Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Gly Glu Pro Gly Asp Arg
Asp Pro Asp Ala Val Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val
Thr Val Ser Ser 115 120138PRThomo sapiens 13Gly Gly Thr Phe Ser Ser
Tyr Ala1 5148PRThomo sapiens 14Ile Ile Arg Phe Leu Gly Lys Thr1
51514PRThomo sapiens 15Ala Gly Glu Pro Gly Asp Arg Asp Pro Asp Ala
Val Asp Ile1 5 1016364DNAhomo sapiens 16caggtccagc tggtgcagtc
tggggctgag gtgatgaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttccggagg
caccttccgc agctatgcta tcagttgggt gcgacaggcc 120cctggacaag
ggcttgagtg gatgggaagg atcatcgttt tccttggtaa aacaaactac
180gcacagaagt tccagggcag agtcacgctt accgcggaca aatccacgac
cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtac gggggaacct 300ggggctcggg accccgacgc ttttgatatc
tggggccaag ggacaatggt caccgtctct 360tcag 36417121PRThomo sapiens
17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Arg Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Ile Val Phe Leu Gly Lys Thr Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Gly Glu Pro Gly Ala Arg Asp Pro
Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val
Ser Ser 115 120188PRThomo sapiens 18Gly Gly Thr Phe Arg Ser Tyr
Ala1 5198PRThomo sapiens 19Ile Ile Val Phe Leu Gly Lys Thr1
52014PRThomo sapiens 20Thr Gly Glu Pro Gly Ala Arg Asp Pro Asp Ala
Phe Asp Ile1 5 1021364DNAhomo sapiens 21caggtccagc tggtgcagtc
tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagc cttccggagg
caccttcagg agctacgcta tcagctgggt acgacaggcc 120cctggacaag
ggcttgagtg gatgggaagg atcatcgttt tccttggtaa agtaaactac
180gcacagaggt ttcagggcag agtcacgctt accgcggaca aatccacgac
cacagcctac 240atggagctga gcagcctgag atctgaggac acggccgtgt
attactgtac gggggaacct 300ggggctcggg accccgacgc ttttgatatc
tggggccaag ggacaatggt caccgtctct 360tcag 36422121PRThomo sapiens
22Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Pro Ser Gly Gly Thr Phe Arg Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Arg Ile Ile Val Phe Leu Gly Lys Val Asn Tyr Ala
Gln Arg Phe 50 55 60Gln Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr
Thr Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Thr Gly Glu Pro Gly Ala Arg Asp Pro
Asp Ala Phe Asp Ile Trp Gly 100 105 110Gln Gly Thr Met Val Thr Val
Ser Ser 115 120238PRThomo sapiens 23Gly Gly Thr Phe Arg Ser Tyr
Ala1 5248PRThomo sapiens 24Ile Ile Val Phe Leu Gly Lys Val1
52514PRThomo sapiens 25Thr Gly Glu Pro Gly Ala Arg Asp Pro Asp Ala
Phe Asp Ile1 5 1026321DNAhomo sapiens 26gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
gggtattcgc agctggttag cctggtatca gcagaaacca 120gagaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataatagtt
acccgctcac tttcggcgga 300gggaccaagg tggagatcaa a 32127107PRThomo
sapiens 27Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105286PRThomo sapiens 28Gly Gly Ile Arg Ser
Trp1 5293PRThomo sapiens 29Ala Ala Ser1309PRThomo sapiens 30Gln Gln
Tyr Asn Ser Tyr Pro Leu Thr1 531322DNAhomo sapiens 31gacatccaga
tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc
gggcgagtca gggtattcgc agctggttag cctggtatca gcagaaacca
120gagaaagccc ctaagtccct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctgcagcct 240gaagattttg caacttatta ctgccaacag
tataatagtt acccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac
32232107PRThomo sapiens 32Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Gly Ile Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105336PRThomo sapiens 33Gln Gly
Ile Arg Ser Trp1 5343PRThomo sapiens 34Ala Ala Ser1359PRThomo
sapiens 35Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1 536322DNAhomo
sapiens 36gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgtc gggcgagtca gggtattcgc agctggttag cctggtatca
gcagaaacca 120gagaaagccc ctaagtccct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagat
ttcactctca ccatcagcag cctgcagcct 240gaagattttg caacttatta
ctgccaacag tataatagtt acccgctcac tttcggcgga 300gggaccaagg
tggagatcaa ac 32237107PRThomo sapiens 37Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln
Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90
95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105386PRThomo
sapiens 38Gln Gly Ile Arg Ser Trp1 5393PRThomo sapiens 39Ala Ala
Ser1409PRThomo sapiens 40Gln Gln Tyr Asn Ser Tyr Pro Leu Thr1
541322DNAhomo sapiens 41gacatccaga tgacccagtc tccatcctca ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca gggtattcgc agctggttag
cctggtatca gcagaaacca 120gagaaagccc ctaagtccct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg gcagtggatc
tgggacagat ttcactctca ccatcagcag cctgcagcct 240gaagattttg
caacttatta ctgccaacag tataataatt atccgctcac tttcggcgga
300gggaccaagg tggagatcaa ac 32242107PRThomo sapiens 42Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Ser Trp 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40
45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn
Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105436PRThomo sapiens 43Gln Gly Ile Arg Ser Trp1 5443PRThomo
sapiens 44Ala Ala Ser1459PRThomo sapiens 45Gln Gln Tyr Asn Asn Tyr
Pro Leu Thr1 546322DNAhomo sapiens 46gacatccaga tgacccagtc
tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgtc gggcgagtca
gggtattcgc agctggttag cctggtatca gcagaaacca 120gagaaagccc
ctaagtccct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca
180aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag
cctgcagcct 240gaagattttg caacttatta ctgccaacag tataataatt
atccgctcac tttcggcgga 300gggaccaagg tggagatcaa ac 32247107PRThomo
sapiens 47Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Arg Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro
Lys Ser Leu Ile 35 40 45Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Tyr Asn Asn Tyr Pro Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105486PRThomo sapiens 48Gln Gly Ile Arg Ser
Trp1 5493PRThomo sapiens 49Ala Ala Ser1509PRThomo sapiens 50Gln Gln
Tyr Asn Asn Tyr Pro Leu Thr1 551300PRThomo sapiens 51Met Ala Asn
Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys1 5 10 15Arg Leu
Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu Val 20 25 30Leu
Ile Leu Val Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40
45Gln Trp Ser Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu
50 55 60Ala Arg Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His
Val65 70 75 80Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe
Ile Ser Lys 85 90 95His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro
Leu Met Lys Leu 100 105 110Gly Thr Gln Thr Val Pro Cys Asn Lys Ile
Leu Leu Trp Ser Arg Ile 115 120 125Lys Asp Leu Ala His Gln Phe Thr
Gln Val Gln Arg Asp Met Phe Thr 130 135 140Leu Glu Asp Thr Leu Leu
Gly Tyr Leu Ala Asp Asp Leu Thr Trp Cys145 150 155 160Gly Glu Phe
Asn Thr Ser Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175Arg
Lys Asp Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185
190Ser Arg Arg Phe Ala Glu Ala Ala Cys Gly Val Val His Val Met Leu
195 200 205Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe
Gly Ser 210 215 220Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln
Thr Leu Glu Ala225 230 235 240Trp Val Ile His Gly Gly Arg Glu Asp
Ser Arg Asp Leu Cys Gln Asp 245 250 255Pro Thr Ile Lys Glu Leu Glu
Ser Ile Ile Ser Lys Arg Asn Ile Gln 260 265 270Phe Ser Cys Lys Asn
Ile Tyr Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285Lys Asn Pro
Glu Asp Ser Ser Cys Thr Ser Glu Ile 290 295 30052300PRTHomo sapiens
52Met Ala Asn Cys Glu Phe Ser Pro Val Ser Gly Asp Lys Pro Cys Cys1
5 10 15Arg Leu Ser Arg Arg Ala Gln Leu Cys Leu Gly Val Ser Ile Leu
Val 20 25 30Leu Ile Leu Val
Val Val Leu Ala Val Val Val Pro Arg Trp Arg Gln 35 40 45Gln Trp Ser
Gly Pro Gly Thr Thr Lys Arg Phe Pro Glu Thr Val Leu 50 55 60Ala Arg
Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val65 70 75
80Asp Cys Gln Ser Val Trp Asp Ala Phe Lys Gly Ala Phe Ile Ser Lys
85 90 95His Pro Cys Asn Ile Thr Glu Glu Asp Tyr Gln Pro Leu Met Lys
Leu 100 105 110Gly Thr Gln Thr Val Pro Cys Asn Lys Ile Leu Leu Trp
Ser Arg Ile 115 120 125Lys Asp Leu Ala His Gln Phe Thr Gln Val Gln
Arg Asp Met Phe Thr 130 135 140Leu Glu Asp Thr Leu Leu Gly Tyr Leu
Ala Asp Asp Leu Thr Trp Cys145 150 155 160Gly Glu Phe Asn Thr Ser
Lys Ile Asn Tyr Gln Ser Cys Pro Asp Trp 165 170 175Arg Lys Asp Cys
Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val 180 185 190Ser Arg
Arg Phe Ala Glu Ala Ala Cys Asp Val Val His Val Met Leu 195 200
205Asn Gly Ser Arg Ser Lys Ile Phe Asp Lys Asn Ser Thr Phe Gly Ser
210 215 220Val Glu Val His Asn Leu Gln Pro Glu Lys Val Gln Thr Leu
Glu Ala225 230 235 240Trp Val Ile His Gly Gly Arg Glu Asp Ser Arg
Asp Leu Cys Gln Asp 245 250 255Pro Thr Ile Lys Glu Leu Glu Ser Ile
Ile Ser Lys Arg Asn Ile Gln 260 265 270Phe Ser Cys Lys Asn Ile Tyr
Arg Pro Asp Lys Phe Leu Gln Cys Val 275 280 285Lys Asn Pro Glu Asp
Ser Ser Cys Thr Ser Glu Ile 290 295 300534PRTArtificial
SequenceSynthetic peptide 53Cys Pro Pro Cys1549PRTArtificial
SequenceSynthetic peptide 54Tyr Pro Tyr Asp Val Pro Asp Tyr Ala1
5556PRTArtificial SequenceSynthetic peptide 55His His His His His
His1 5
* * * * *
References